Cyclic polypeptides for the treatment of heart failure

ABSTRACT

The invention provides a cyclic polypeptide of Formula I (SEQ ID NO: 1): 
       X1-R-X3-X4-L-S-X7-X8-X9-X10-X11-X12-X13  I
         or an amide, an ester or a salt thereof, or a bioconjugate thereof, wherein X1, X3, X4, X7, X8, X9, X10, X11, X12 and X13 are defined herein. The polypeptides are agonist of the APJ receptor. The invention also relates to a method for manufacturing the polypeptides of the invention or bioconjugates thereof, and their therapeutic uses such as treatment or prevention of acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral arterial disease, cerebrovascular accidents, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn) and preeclampsia. The present invention further provides a combination of pharmacologically active agents and a pharmaceutical composition.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 29, 2014, isnamed PAT055418-US-NP_SL.txt and is 44,387 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel compositions comprising modified peptideand polypeptide sequences designed to treat cardiovascular disease insubjects to whom they are administered, and which exhibit greaterresistance to degradation, and equivalent or greater bioactivity thantheir wild type counterparts. The invention also relates to methods ofmaking said compositions and using said compositions as pharmaceuticallyactive agents to treat cardiovascular disease.

BACKGROUND OF THE INVENTION

The incidence of heart failure in the Western world is approximately1/100 adults after 65 yrs of age. The most common pathology is a chronicdeficit in cardiac contractility and, thereby, cardiac output, i.e., theeffective volume of blood expelled by either ventricle of the heart overtime. Patients with chronic heart failure can have acute episodes ofdecompensation, i.e., failure of the heart to maintain adequate bloodcirculation, where cardiac contractility declines further. There are˜500K hospitalizations per year for “acute decompensated heart failure”(ADHF) in the USA alone.

Current therapies for ADHF include diuretics, vasodilators, andinotropes, which directly increase cardiac contractility. Currentintravenous inotropes (dobutamine, dopamine, milrinone, levosimendan)are used in the acute setting, despite their association with adverseevents such as arrhythmia and increased long-term mortality. Theseliabilities have prevented their application in chronic heart failure.Digoxin is an oral inotrope, but is limited by a narrow therapeuticindex, increased arrhythmogenic potential and contraindication in renalinsufficiency.

A therapy for heart failure that increases cardiac contractility withoutarrhythmogenic or mortality liabilities is urgently needed for ADHF, butcould also address the enormous unmet medical need in chronic heartfailure.

Apelin is the endogenous ligand for the previously orphanG-protein-coupled receptor (GPCR), APJ, also referred to as apelinreceptor, angiotension-like-1 receptor, angiotension II-like-1 receptor,and the like. The apelin/APJ pathway is widely expressed in thecardiovascular system and apelin has shown major beneficialcardiovascular effects in preclinical models. Acute apelinadministration in humans causes peripheral and coronary vasodilatationand increases cardiac output (Circulation. 2010; 121:1818-1827). As aresult, APJ agonism is emerging as an important therapeutic target forpatients with heart failure. Activation of the apelin receptor APJ isthought to increase cardiac contractility and provide cardioprotection,without the liabilities of current therapies. However, the nativeapelins exhibit a very short half life and duration of action in vivo.The very short half life is a recognized major difficulty with thedelivery of such therapeutic endogenous peptides due to rapid serumclearance and proteolytic degradation via the action of peptidases.

One way which has been currently used to overcome this disadvantage isto administer large dosage of therapeutic peptide of interest to thepatient so that even if some therapeutic peptide is degraded, enoughremains to be therapeutically effective. However, this method isuncomfortable to patients. Since most therapeutic peptides cannot beadministered orally, the therapeutic peptide would have to be eitherconstantly infused, frequently infused by intravenous injection oradministered frequently by the inconvenient route of subcutaneousinjections. The need for frequent administration also results in manypotential peptide therapeutics having an unacceptable high projectedcost of treatment. The presence of large amounts of degraded peptide mayalso generate undesired side effects.

Discomfort in administration and high costs are two reasons why mosttherapeutic peptides with attractive bioactivity profiles may not bedeveloped as drug candidates.

Therefore, one approach to prolong half-life of peptides is to modifythe therapeutic peptides in such a way that their degradation is sloweddown while still maintaining biological activity.

It is therefore desirable to identify peptides and polypeptides thatmimic the function of apelin, but have increased half-life anddemonstrate equivalent or greater bioactivity than the naturallyoccurring apelin. Furthermore, it is desirable to identify apelin analogpeptides and polypeptides which exhibit increased conformationalconstraints, i.e., the ability to achieve and maintain an activeconformational state such that the peptides and polypeptides caninteract with their receptors and/or other pathway targets without theneed for additional folded or repositioning. Additional approachesincludes reducing the rate of clearance by conjugating the peptides tomolecules that prevent their elimination through kidney.

There is thus a need for modified therapeutic peptides with increasedhalf-life in order to provide longer duration of action in vivo, whilemaintaining low toxicity yet retaining the therapeutic advantages of themodified peptides.

SUMMARY OF THE INVENTION

This invention is directed to overcoming the problem of peptidedegradation in the body by modifying the therapeutic peptide orpolypeptide of interest, i.e. APJ agonists.

The aim of the present invention is to provide novel peptides andpolypeptides or bioconjugate thereof, which are useful as APJ agonists,and which also possess at least one of the following improvements overwild type apelin and other known apelin analogs: increased half-life;greater immunity to degradation upon administration and/or uponsolubilization; and increased conformational constraints, all whileexhibiting the same or greater biological activity as wild type apelin.The peptides and polypeptides of this invention, or bioconjugatesthereof, are thus particularly useful for the treatment or prevention ofcardiovascular diseases such as heart failure, disorders and conditionsassociated with heart failure, and disorders and conditions responsiveto the activation of APJ receptor activity.

In one embodiment, the peptides and polypeptides of the invention, orbioconjugates thereof, are particularly useful for the treatment orprevention of a disorder or condition associated with heart failure, ora disorder responsive to the activation (or agonism) of the APJ receptoractivity. In another embodiment, the peptides and polypeptides of theinvention, or bioconjugates thereof, are useful in the treatment ofacute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

The invention pertains to the peptides and polypeptides, orbioconjugates thereof, pharmaceutical compositions, and methods ofmanufacture and use thereof, as described herein. Examples of peptidesand polypeptides of the invention include the peptides and polypeptidesaccording to any one of Formulae I to IV, or an amide, an ester or asalt thereof, as well as any peptides or polypeptides specificallylisted herein, and bioconjugates thereof, including but not limited tothe experimental examples.

The invention therefore provides a peptide or a polypeptide formula (I)(SEQ ID NO:1):

X1-R-X3-X4-L-S-X7-X8-X9-X10-X11-X12-X13  I

wherein:X1 is the N-terminus of the polypeptide and is either absent or is Q, Aor pE or X1 is selected from C, c, hC, D-hC; wherein the side chain ofC, c, hC or D-hC form a disulfide bond with the side chain of X7;X3 is P or X3 is selected from C, c, hC and D-hC; wherein the side chainof C, c, hC or D-hC forms a disulfide bond with the side chain of X7;X4 is R or X4 is selected from C, c, hC and D-hC; wherein the side chainof C, c, hC or D-hC forms a disulfide bond with the side chain of X7;wherein only one of X1, X3 and X4 is a sulfur contain amino-acidselected from C, c, hC and D-hC;X7 is C, c, hC or D-hC; and the side chain of X7 forms a disulfide bondwith the side chain of C, c, hC or D-hC of either X1, X3 or X4;

X8 is K or F;

X9 is G, A, a or absent;X10 is P or absent;

X11 is D-Nle, Nle, M or f; and

X12 is absent or P, f, a, D-Nva or D-Abu;X13 is the C-terminus and is absent or is selected from (N-Me)F, F, f,a, y and Nal; wherein:

Nle is L-norleucine; D-Nle is D-norleucine; D-hC is D-homocysteine hC isL-homocysteine; Nal is L-naphathaline; D-Nva is D-norvaline;

D-Abu is D-2-aminobutyric acid;pE is L-pyroglutamic acid;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereof.

As further explained herein, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. When the oneletter abbreviation is a capital letter, it refers to the L-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe D-amino acid. Alternatively, a D-amino acid can be represented witha D-letter in front of the abbreviation letter(s). i.e., when precededwith “D,” the amino acid is a D-amino acid.

Any of the above-listed amino acid residues of Formula I, or its relatedformulae described herein, e.g., Formulae I to IV, may be substituted ina conservative fashion, provided the peptide or polypeptide of theinvention still retains functional activity and structural properties(e.g., half-life extension, protection from degradation, conformationalconstraint). Principle and examples of permissible conservative aminoacid substitutions are further explained herein.

The invention further provides a bioconjugate or a multimer thereof,comprising:

-   -   a. a peptide or a polypeptide of anyone of Formulae I to IV,    -   b. a half-life extending moiety;    -   wherein said peptide or polypeptide and said half life are        covalently linked or fused, optionally via a linker.

“Optionally via a linker” means that the peptide of polypeptideaccording to Formulae I-IV is covalently linked or fused to thehalf-life extending moiety directly (i.e. with no linker) or iscovalently linked or fused to the half-life extending moiety via alinker.

The half-life extending moiety of the invention can be covalently fused,attached, linked or conjugated to a peptide or polypeptide analog. Ahalf-life extending moiety can be, for example, a polymer, such aspolyethylene glycol (PEG), a cholesterol group, a carbohydrate oroligosaccharide; a fatty acid or any natural or synthetic protein,polypeptide or peptide that binds to a salvage receptor. Preferably, thehalf-life extending moiety is covalently linked, optionally via alinker, to plasma protein (albumin and immunoglobulin) with long serumhalf-lives. In other embodiment, the half-life extending moiety is analbumin binding residue. An “Albumin binding residue” as used hereinmeans a residue which binds non-covalently to human serum albumin. Inone embodiment the albumin binding residue is a lipophilic residue. Inanother embodiment, the albumin binding residue is negatively charged atphysiological pH. An albumin binding residue typically comprises acarboxylic acid which can be negatively charged. Examples of albuminbinding residue includes fatty acids. In other embodiment, the half-lifeextending moiety is an IgG constant domain or fragment thereof (e.g.,the Fc region), Human Serum Albumin (HSA), or an albumin-bindingpolypeptides or residue such as for example a fatty acid. Preferably,the half-life extending moiety portion of the bioconjugate is a humanserum albumin or an Fc region. Most preferably, the half-life extendingmoiety portion of the bioconjugate is an Fc region.

The half-life extending moiety is attached in such a way so as enhance,and/or not to interfere with, the biological function of the constituentportions of the bio-conjugates of the invention, e.g., the peptide orpolypeptide of the invention (Formulae I-IV). In some embodiments, thepolypeptide of the invention can be fused to a half-life extendingmoiety, optionally via a linker. The half-life extending moiety can be aprotein such as an IgG constant domain or fragment thereof (e.g., the Fcregion), Human Serum Albumin (HSA), or albumin-binding polypeptides orresidue (e.g. a fatty acid). Such proteins disclosed herein can alsoform multimers.

In some embodiments, the half-life extending moiety (e.g., HSA, Fc,fatty acid etc.) is covalently linked or fused to the N-terminus of thepeptide or polypeptide of any one of Formulae I-IV. In otherembodiments, the half-life extending moiety (e.g., HSA, Fc, fatty acidetc.) is covalently linked or fused to C-terminus of the peptide orpolypeptide of any one of Formulae I to IV of the invention.

The polypeptides of the invention, or bioconjugates thereof, viaactivation of the APJ receptor, have utility in the treatment of acutedecompensated heart failure (ADHF), chronic heart failure, pulmonaryhypertension, atrial fibrillation, Brugada syndrome, ventriculartachycardia, atherosclerosis, hypertension, restenosis, ischemiccardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia,water retention, diabetes (including gestational diabetes), obesity,peripheral arterial disease, cerebrovascular accidents, transientischemic attacks, traumatic brain injuries, amyotrophic lateralsclerosis, burn injuries (including sunburn) and preeclampsia.

In a preferred embodiment the polypeptides of the invention, orbioconjugates thereof, are useful in the treatment of acutedecompensated heart failure (ADHF).

In another embodiment, the invention pertains to a method for treatingdisorder or disease responsive to the activation of the APJ receptor, ina subject in need of such treatment, comprising: administering to thesubject an effective amount of a polypeptide according to any one ofFormulae I to IV, or an amide, an ester of a salt thereof, or abioconjugate thereof, such that the disorder or disease responsive tothe activation of the APJ receptor in the subject is treated.

In yet another embodiment, the invention pertains to pharmaceuticalcompositions, comprising a polypeptide according to any one of FormulaeI to IV, or an amide, an ester or salt thereof, or a bioconjugatethereof, and one or more pharmaceutically acceptable carriers.

In still another embodiment, the invention pertains to combinationsincluding, a polypeptide according to any one of Formulae I to IV, or anamide, an ester or a salt thereof, or a bioconjugate thereof, andpharmaceutical combinations of one or more therapeutically activeagents.

In another embodiment, the invention pertains to a method for activationof the APJ receptor in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of apolypeptide according to any one of Formulae I to IV, or an amide, anester or a salt thereof or a bioconjugate thereof.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of interpreting this specification, the followingdefinitions will apply unless specified otherwise and wheneverappropriate, terms used in the singular will also include the plural andvice versa.

As used herein, “disorders or diseases responsive to the modulation ofthe APJ receptor,” “disorders and conditions responsive to themodulation of the APJ,” “disorders and conditions responsive to themodulation of APJ receptor activity,” “disorders responsive to theactivation (or agonism) of the APJ receptor activity,” and like termsinclude acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

As used herein, “Activation of APJ receptor activity,” or “Activation ofthe APJ receptor,” refers to an increase in the APJ receptor activity.The activation of the APJ receptor activity is also referred to as“agonism” of the APJ receptor, e.g., by administration of the peptidesand polypeptides of the invention.

As used herein, the terms “polypeptide” and “peptide” are usedinterchangeably to refer to two or more amino acids linked together.Except for the abbreviations for the uncommon or unnatural amino acidsset forth in Table 1 below, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D”, the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the D-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe L-amino acid. Groups or strings or amino acid abbreviations are usedto represent peptides. Peptides are indicated with the N-terminus on theleft and the sequence is written from the N-terminus to the C-terminus.

Peptides of the invention contain non-natural amino acids (i.e.,compounds that do not occur in nature) and other amino acid analogs asare known in the art may alternatively be employed.

Certain non-natural amino acids can be introduced by the technologydescribed in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wangand Schultz, Science 301:964-967, 2003; Wang et al., Science292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in U.S.Pat. No. 7,083,970. Briefly, some of these expression systems involvesite-directed mutagenesis to introduce a nonsense codon, such as anamber TAG, into the open reading frame encoding a polypeptide of theinvention. Such expression vectors are then introduced into a host thatcan utilize a tRNA specific for the introduced nonsense codon andcharged with the non-natural amino acid of choice. Particularnon-natural amino acids that are beneficial for purpose of conjugatingmoieties to the polypeptides of the invention include those withacetylene and azido side chains.

One or more of the natural or un-natural amino acids in a peptide of theinvention may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Saidmodifications may be done in a site-specific or non-site-specificmanner. In a preferred embodiment, the modifications of the peptide leadto a more stable peptide (e.g., one exhibiting greater half-life invivo). These modifications may include the incorporation of additionalD-amino acids, etc. None of the modifications should substantiallyinterfere with the desired biological activity of the peptide, but suchmodifications may confer desirable properties, e.g., enhanced biologicalactivity, on the peptide.

Said modifications enhance the biological properties of the proteins ofthe invention relative to the wild-type proteins, as well as, in somecases, serving as points of attachment for, e.g., labels and proteinhalf-life extension agents, and for purposes of affixing said variantsto the surface of a solid support.

In certain embodiments, such modifications, e.g., site-specificmodifications, are used to attach conjugates, e.g., PEG groups topolypeptides, and/or peptides of the invention, for purposes of, e.g.,extending half-life or otherwise improving the biological properties ofsaid polypeptides, and/or peptides. Said techniques are describedfurther herein.

In other embodiments, such modifications, e.g., site-specificmodifications, are used to attach other polymers and small molecules andrecombinant protein sequences that extend half-life of the polypeptideof the invention. One such embodiment includes the attachment of fattyacids or specific albumin binding compounds to polypeptides, and/orpeptides. In other embodiments, the modifications are made at aparticular amino acid type and may be attached at one or more sites onthe polypeptides.

In other embodiments, such modifications, e.g., site-specificmodifications, are used as means of attachment for the production ofwild-type and/or variant multimers, e.g., dimers (homodimers orheterodimers) or trimers or tetramers. These multimeric proteinmolecules may additionally have groups such as PEG, sugars, and/orPEG-cholesterol conjugates attached or be fused either amino-terminallyor carboxy-terminally to other proteins such as Fc, Human Serum Albumin(HSA), etc.

In other embodiments, such site-specific modifications are used toproduce proteins, polypeptides and/or peptides wherein the position ofthe site-specifically incorporated pyrrolysine or pyrrolysine analogueor non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe)allows for controlled orientation and attachment of such proteins,polypeptides and/or peptides onto a surface of a solid support or tohave groups such as PEG, sugars and/or PEG-cholesterol conjugatesattached.

In other embodiments, such site-specific modifications are used tosite-specifically cross-link proteins, polypeptides and/or peptidesthereby forming hetero-oligomers including, but not limited to,heterodimers and heterotrimers. In other embodiments, such site-specificmodifications are used to site-specifically cross-link proteins,polypeptides and/or peptides thereby forming protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates. In other embodiments, a site specificmodification may include a branching point to allow more than one typeof molecule to be attached at a single site of a protein, polypeptide orpeptide.

In other embodiments, the modifications listed herein can be done in anon-site-specific manner and result in protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates of the invention.

In some embodiments, the present invention provides complexes whichcomprise at least one peptide or polypeptide of any one of Formulae I-IVbound to an antibody, such as an antibody which specifically binds apeptide or polypeptide as disclosed herein.

One of ordinary skill in the art will appreciate that various amino acidsubstitutions, e.g, conservative amino acid substitutions, may be madein the sequence of any of the polypeptides described herein, withoutnecessarily decreasing its activity. As used herein, “amino acidcommonly used as a substitute thereof” includes conservativesubstitutions (i.e., substitutions with amino acids of comparablechemical characteristics). For the purposes of conservativesubstitution, the non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophanand methionine. The polar (hydrophilic), neutral amino acids includeserine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of amino acid substitutions includesubstituting an L-amino acid for its corresponding D-amino acid,substituting cysteine for homocysteine or other non natural amino acidshaving a thiol-containing side chain, substituting a lysine forhomolysine, diaminobutyric acid, diaminopropionic acid, ornithine orother non natural amino acids having an amino containing side chain, orsubstituting an alanine for norvaline or the like.

The term “amino acid,” as used herein, refers to naturally occurringamino acids, unnatural amino acids, amino acid analogues and amino acidmimetics that function in a manner similar to the naturally occurringamino acids, all in their D and L stereoisomers if their structureallows such stereoisomeric forms. Amino acids are referred to herein byeither their name, their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “naturally occurring” refers to materials which are found innature and are not manipulated by man. Similarly, “non-naturallyoccurring,” “un-natural,” and the like, as used herein, refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man. When used in connection with aminoacids, the term “naturally occurring” refers to the 20 conventionalamino acids (i.e., alanine (A or Ala), cysteine (C or Cys), asparticacid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe),glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine(K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N orAsn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg),serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan(W or Trp), and tyrosine (Y or Tyr)).

The terms “non-natural amino acid” and “unnatural amino acid,” as usedherein, are interchangeably intended to represent amino acid structuresthat cannot be generated biosynthetically in any organism usingunmodified or modified genes from any organism, whether the same ordifferent. The terms refer to an amino acid residue that is not presentin the naturally occurring (wild-type) apelin protein sequence or thesequences of the present invention. These include, but are not limitedto, modified amino acids and/or amino acid analogues that are not one ofthe 20 naturally occurring amino acids, selenocysteine, pyrrolysine(Pyl), or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCTpatent publication WO2010/48582). Such non-natural amino acid residuescan be introduced by substitution of naturally occurring amino acids,and/or by insertion of non-natural amino acids into the naturallyoccurring (wild-type) Apelin protein sequence or the sequences of theinvention. The non-natural amino acid residue also can be incorporatedsuch that a desired functionality is imparted to the apelin molecule,for example, the ability to link a functional moiety (e.g., PEG). Whenused in connection with amino acids, the symbol “U” shall mean“non-natural amino acid” and “unnatural amino acid,” as used herein.

In addition, it is understood that such “unnatural amino acids” requirea modified tRNA and a modified tRNA synthetase (RS) for incorporationinto a protein. These “selected” orthogonal tRNA/RS pairs are generatedby a selection process as developed by Schultz et al. or by random ortargeted mutation. As way of example, pyrroline-carboxy-lysine is a“natural amino acid” as it is generated biosynthetically by genestransferred from one organism into the host cells and as it isincorporated into proteins by using natural tRNA and tRNA synthetasegenes, while p-aminophenylalanine (See, Generation of a bacterium with a21 amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang L,Martin A B, King D S, Horn D M, Schultz P G. J Am Chem Soc. 2003 Jan.29; 125(4):935-9) is an “unnatural amino acid” because, althoughgenerated biosynthetically, it is incorporated into proteins by a“selected” orthogonal tRNA/tRNA synthetase pair.

Modified encoded amino acids include, but are not limited to,hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidinecarboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminoproprionic acid, N-ethylglycine, N-methylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.The term “amino acid” also includes naturally occurring amino acids thatare metabolites in certain organisms but are not encoded by the geneticcode for incorporation into proteins. Such amino acids include, but arenot limited to, ornithine, D-ornithine, and D-arginine.

The term “amino acid analogue,” as used herein, refers to compounds thathave the same basic chemical structure as a naturally occurring aminoacid, by way of example only, an α-carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group. Amino acid analoguesinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or their C-terminal carboxy group,their N-terminal amino group and/or their side-chain functional groupsare chemically modified. Such analogues include, but are not limited to,methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteinesulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alaninecarboxamide, homoserine, norleucine, and methionine methyl sulfonium.

TABLE 1 Un-natural or non-natural amino acids as described in theinvention: Symbol Name Structure Nva or nva (D-Nva) L-Norvaline orD-Norvaline

1-Nal 1-Naphthylalanine

2-Nal 2-Naphthylalanine

Nle or nle (D-Nle) L-Norleucine or D-Norleucine

pE Pyroglutamic acid

Abu or abu (D-Abu) 2-amino-butyric acid

O2Oc 8-Amino-3,6-dioxaoctanoic acid

Nal refers to both 1-Naphthylalanine and 2-Naphthylalanine, preferably2-naphthylalanine.

As used herein the term “amide” refers to an amide derivative of thecarboxylic acid group at the C-terminus (e.g. —C(O)NH₂, —C(O)NH—C₁₋₆alkyl, —C(O)NH—C₁₋₂alkylphenyl, —C(O)NH—NHBn, —C(O)-4 phenoxypiperidineor —C(O)N(C₁₋₆ alkyl)₂).

The term “amide” also refers to derivatives of the amino group at theN-terminus (e.g. —NHC(O)C₁₋₁₆alkyl, —NHC(O)(CH₂)_(n)Ph (n is an integerof 1 to 6), —NHC(O)(CH₂)₂CO₂H, 4-Cl-Ph-(CH₂)₃C(O)NH—,C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—,C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—;C₁₅H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)NH—, Ph-CH₂CH₂NHC(O)—NH— orCH₃(OCH₂CH₂)_(m)C(O)NH— (m is an integer of 1 to 12).

As used herein, the term “ester” refers to an ester derivative of thecarboxylic acid group at the C-terminus (e.g —COOR) form wherein R ofthe ester refers to C₁₋₆ alkyl groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, etc., C₃₋₈ cycloalkyl groups such as cyclopentyl,cyclohexyl, etc., C₆₋₁₀ aryl groups such as phenyl, α-naphthyl, etc.,C₆₋₁₀ aryl-C₁₋₆ alkyl groups, for example phenyl-C₁₋₂ alkyl groups suchas benzyl, phenethyl, benzhydryl, etc., and α-naphthyl-C₁₋₂ alkyl groupssuch as α-naphthylmethyl and the like. Mention may also be made ofpivaloyloxymethyl ester and the like, which are commonly used as estersfor oral administration. When the polypeptides of the invention possessadditional carboxyl or carboxylate groups in positions other than the Cterminus, those polypeptides in which such groups are amidated oresterified also fall under the category of the polypeptide of theinvention. In such cases, the esters may for example be the same kindsof esters as the C-terminal esters mentioned above.

The term alkyl refers to a fully saturated branched or unbranched (orstraight chain or linear) hydrocarbon moiety, comprising 1 to 20 carbonatoms. Preferably the alkyl comprises 1 to 7 carbon atoms, and morepreferably 1 to 4 carbon atoms.

The term aryl refers to monocyclic or bicyclic aromatic hydrocarbongroups having 6-10 carbon atoms in the ring portion. Representativeexamples of aryl are phenyl or naphthyl.

The term heteroaryl includes monocyclic or bicyclic heteroaryl,containing from 5-10 ring members selected from carbon atoms and 1 to 5heteroatoms, and each heteroatoms is independently selected from O, N orS wherein S and N may be oxidized to various oxidation states. Forbicyclic heteroaryl system, the system is fully aromatic (i.e. all ringsare aromatic).

The term cycloalkyl refers to saturated or unsaturated but non-aromaticmonocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbonatoms, preferably 3-8, or 3-7 carbon atoms. For bicyclic, and tricycliccycloalkyl system, all rings are non-aromatic.

The term heterocyclyl refers to a saturated or unsaturated non-aromatic(partially unsaturated) ring which is a 4-, 5-, 6-, or 7-memberedmonocyclic, and contains at least one heteroatom selected from O, S andN, where the N and S can also optionally be oxidized to variousoxidation states. In one embodiment, heterocyclyl moiety represents asaturated monocyclic ring containing from 5-7 ring atoms and optionallycontaining a further heteroatom, selected from O, S or N.

The term “APJ” (also referred to as “apelin receptor,”“angiotension-like-1 receptor,” “angiotension II-like-1 receptor,” andthe like) indicates a 380 residue, 7 transmembrane domain, Gi coupledreceptor whose gene is localized on the long arm of chromosome 11 inhumans (NCBI Reference Sequence: NP_005152.1, and encoded by NCBIReference Sequence: NM_005161). APJ was first cloned in 1993 fromgenomic human DNA using degenerate oligonucleotide primers (O'Dowd etal. Gene, 136:355-60, 1993) and shares significant homology withangiotensin II receptor type 1. Despite this homology however,angiotensin II does not bind APJ. Although orphan for many years, theendogenous ligand has been isolated and named apelin (Tatemoto et al.,Biochem Biophys Res Commun 251, 471-6 (1998)).

The term “apelin,” indicates a 77 residue preprotein (NCBI ReferenceSequence: NP_0059109.3, and encoded by NCBI Reference Sequence:NM_017413.3), which gets processed into biologically active forms ofapelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13,apelin-12. The full length mature peptide, referred to as “apelin-36,”comprises 36 amino acids, but the most potent isoform is thepyroglutamated form of a 13mer of apelin (apelin-13), referred to as“Pyr-1-apelin-13 or Pyr¹-apelin-13” Different apelin forms aredescribed, for instance, in U.S. Pat. No. 6,492,324B1.

The term “conjugate” and “bioconjugate” is used interchangeably and isintended to refer to the entity formed as a result of a covalentattachment between a polypeptide of anyone of Formulae I to IV, and ahalf-life extending moiety, optionally via linker. The term “Conjugate”or “bioconjugate” is also intended to include an entity formed as aresult of a fusion between an APJ agonist polypeptide or a polypeptideof Formula I, II, III or IV, and a half life extending moiety.

The term half-life extending moiety can be covalently linked/attached orfused to a peptide or polypeptide analog. A half-life extending moietycan be, for example, a polymer, such as polyethylene glycol (PEG), fattyacid, a cholesterol group, a carbohydrate or olisaccharide; or anynatural or synthetic protein, polypeptide or peptide that binds to asalvage receptor. In other embodiment, the half-life extending moiety isan albumin binding residue. An “Albumin binding residue” as used hereinmeans a residue which binds non-covalently to human serum albumin. Inone embodiment the albumin binding residue is a lipophilic residue. Inanother embodiment, the albumin binding residue is negatively charged atphysiological pH. An albumin binding residue typically comprises acarboxylic acid which can be negatively charged. Examples of albuminbinding residue includes fatty acids. In other embodiment, the half-lifeextending moiety is covalently linked, optionally via a linker, toplasma protein (albumin and immunoglobulin) with long serum half-lives.For example, the half-life extending moiety is an IgG constant domain orfragment thereof (e.g., the Fc region), Human Serum Albumin (HSA), or analbumin-binding polypeptides or residue (e.g a fatty acid). Mostpreferably, the half-life extending moiety portion of the bioconjugateis an Fc region.

The term “increased half-life” or “increase serum half-life” or“extending half-life” is meant the positive change in circulatinghalf-life of a modified biologically active molecule (e.g. apelin 13)relative to its non-modified form (or naked form of the peptide). Serumhalf-life is measured by taking blood samples at various time pointsafter administration of the biologically active molecule, anddetermining the concentration of that molecule in each sample. Measuringthe change in serum concentration with time allows calculation of theserum half-life of a modified molecule (e.g. conjugated molecule). Bycomparing the serum half-life of a modified molecule (e.g. conjugatedmolecule), with an unmodified molecule (e.g. apelin 13), the relativeincrease in serum half-life or t½ may be determined. The increase isdesirably at least about two-fold, but a smaller increase may be useful.

Polypeptides of the Invention:

Various embodiments of the invention are described herein. It will berecognized that features specified in each embodiment may be combinedwith other specified features to provide further embodiments.

In embodiment 1, the invention therefore provides a peptide or apolypeptide formula (I) (SEQ ID NO: 1):

X1-R-X3-X4-L-S-X7-X8-X9-X10-X11-X12-X13  I

wherein:X1 is the N-terminus of the polypeptide and is either absent, Q, A or pEor X1 is selected from C, c, hC, D-hC; wherein the side chain of C, c,hC or D-hC form a disulfide bond with the side chain of X7;X3 is P or X3 is selected from C, c, hC and D-hC; wherein the side chainof C, c, hC or D-hC forms a disulfide bond with the side chain of X7;X4 is R or X4 is selected from C, c, hC and D-hC; wherein the side chainof C, c, hC or D-hC forms a disulfide bond with the side chain of X7;wherein only one of X1, X3 and X4 is a sulfur contain amino-acidselected from C, c, hC and D-hC;X7 is C, c, hC or D-hC; and the side chain of X7 forms a disulfide bondwith the side chain of C, c, hC or D-hC of either X1, X3 or X4;

X8 is K or F;

X9 is G, A or a or absent;X10 is P or absent;

X11 is D-Nle, Nle, M or f; and

X12 is absent or P, f, a, D-Nva or D-Abu;X13 is the C-terminus and is absent or is selected from (N-Me)F, F, f,a, y and Nal; wherein: Nle is L-norleucine;

D-Nle is D-norleucine; D-hC is D-homocysteine hC is L-homocysteine; Nalis L-naphathaline; D-Nva is D-norvaline;

D-Abu is D-2-aminobutyric acid;pE is L-pyroglutamic acid;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In embodiment 2, the invention therefore provides a peptide or apolypeptide of formula (II) (SEQ ID NO: 2):

wherein:X1 is the N-terminus of the polypeptide and is either absent, Q, A orpE;

X4 is C, c, hC or D-hC;

X7 is C, c, hC or D-hC; and the side chain of X7 forms a disulfide bondwith the side chain of

X4; X8 is K or F;

X9 is G, A, a or absent;X10 is P or absent;

X11 is D-Nle, M, Nle or f; and

X12 is absent or is selected from P, f, a, D-Nva and D-Abu;X13 is the C-terminus and is absent or is selected from F, (N-Me)F, f,a, y and Nal; or an amide, an ester or a salt of the polypeptide, or apolypeptide substantially equivalent thereto.

In embodiment 2A, the invention therefore provides a peptide or apolypeptide of formula (II) (SEQ ID NO: 3):

wherein:X1 is the N-terminus of the polypeptide and is either absent or pE;

X4 is C, c, hC or D-hC;

X7 is C, c, hC or D-hC; and the side chain of X7 forms a disulfide bondwith the side chain of

X4; X8 is K or F;

X9 is G, A, a or absent;X10 is P or absent;

X11 is D-Nle, Nle or f; and

X12 is absent or is selected from P, f, a, D-Nva and D-Abu;X13 is the C-terminus and is absent or is selected from F, (N-Me)F, f,a, y and Nal; or an amide, an ester or a salt of the polypeptide, or apolypeptide substantially equivalent thereto.

In embodiment 3, the invention therefore provides a peptide or apolypeptide of formula III (SEQ ID NO: 4):

whereinX1 is the N-terminus of the polypeptide and is selected from C, c, hCand D-hC;X7 is C, c, hC or D-hC; wherein the side chain of X7 forms a disulfidebond with the side chain of X1;

X8 is K or F;

X9 is G, A, a or absent;X10 is P or absent;

X11 is D-Nle, Nle, M or f; and

X12 is absent or is selected from P, f, a, D-Nva and D-Abu;X13 is the C-terminus and is absent or is selected from (N-Me)F, F, f,a, y and Nal;or an amide, an ester or a salt of the polypeptide.

In embodiment 3A, the invention pertains to polypeptides of Formula IIIwherein X11 is D-Nle, Nle or f, or an amide, an ester or a salt thereof.

In embodiment 4, the invention therefore provides a peptide or apolypeptide of Formula IV (SEQ ID NO: 5):

wherein:X1 is the N-terminus of the polypeptide and is either absent, Q, A orpE;X3 is C, c, hC or D-hC; wherein the side chain of C, c, hC or D-hC;X7 is C, c, hC or D-hC; and the side chain of X7 form a disulfide bondwith the side chain of C, c, hC or D-hC of X3;

X8 is K or F;

X9 is G, A, a or absent;X10 is P or absent;

X11 is D-Nle, Nle, M or f; and

X12 is absent or is selected from P, f, a, D-Nva and D-Abu;X13 is the C-terminus and is absent or is selected from (N-Me)F, F, f,a, y and Nal; or an amide, an ester or a salt of the polypeptide.

In embodiment 4A, the invention therefore provides a peptide or apolypeptide of Formula IV (SEQ ID NO: 6):

wherein:X1 is the N-terminus of the polypeptide and is either absent or pE;X3 is C, c, hC or D-hC; wherein the side chain of C, c, hC or D-hC;X7 is C, c, hC or D-hC; and the side chain of X7 form a disulfide bondwith the side chain of C, c, hC or D-hC of X3;

X8 is K or F;

X9 is G, A, a or absent;X10 is P or absent;

X11 is D-Nle, Nle or f; and

X12 is absent or is selected from P, f, a, D-Nva and D-Abu;X13 is the C-terminus and is absent or is selected from (N-Me)F, F, f,a, y and Nal; or an amide, an ester or a salt of the polypeptide.

In embodiment 5, the invention pertains to a polypeptide according toany one of embodiments 1, 2 and 4 wherein X1 is pE; or an amide, anester or a salt of the polypeptide.

In embodiment 5A, the invention pertains to a polypeptide according toany one of embodiments 1, 2, and 4 wherein X1 is A or Q; or an amide, anester or a salt of the polypeptide. In a particular aspect of thisembodiment, the peptide is chemically linked or fused to a half-lifeextending moiety via it's A or Q N-terminus.

In embodiment 6, the invention pertains to a polypeptide according toany one of embodiments 1, 2, 4 and 4A wherein X1 is absent; or an amide,an ester or a salt of the polypeptide.

In embodiment 7, the invention pertains to a polypeptide according toany one embodiments 1 to 4 and 6 wherein the N-terminus is an amide; ora salt of the polypeptide.

In embodiment 8, the invention pertains to a polypeptide according toembodiment 7 wherein the N-terminus is an amide of Formula —NHR and R isAcetyl, benzoyl, phenacyl, succinyl, octanoyl, 4-phenylbutanoyl,4-Cl-Ph-(CH₂)₃C(O)—, or Ph-CH₂CH₂NHC(O)—; or a salt of the polypeptide.

In embodiment 8A, the invention pertains to a polypeptide of any one ofembodiments 1 to 4 and 7 wherein the N-terminus is an amide of FormulaNHR1 wherein R1 is CH₃C(O)—, CH₃—(O—CH₂CH₂)_(m)—C(O)—,Palmitoyl(O2Oc)_(p), Myristoyl(O2Oc)_(p), Lauroyl(O2Oc)_(p) orPh-CH₂CH₂NHC(O)—; and wherein

p is an integer of 1 to 4;m is an integer of 1 to 12;Lauroyl(O2Oc) is C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—;Myristoyl(O2Oc) is C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—;Palmitoyl(O2Oc) is C₁₅H₃₁C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—; or a saltof the polypeptide. Examples of N-terminus amides have been described inU.S. provisional application No. 61/591,557 (Attorney Docket NumberPAT054961-US-PSP) filed on Jan. 27, 2012, which is hereby incorporatedby reference.

In embodiment 9, the invention pertains to a polypeptide according toany one of embodiments 1 to 8A wherein X13 is F or f; or an amide, anester or a salt of the polypeptide.

In embodiment 10, the invention pertains to a polypeptide according toany one of embodiments 1 to 8A wherein X13 is absent; or an amide, anester or a salt of the polypeptide.

In embodiment 11, the invention pertains to a polypeptide according toany one of embodiments 1-10 wherein X12 is absent; or an amide, andester or a salt of the polypeptide.

In embodiment 12, the invention pertains to a polypeptide according toany one of embodiments 1 to 11 wherein the C-terminus is an amide; or asalt of the polypeptide.

In embodiment 13, the invention pertains to a polypeptide according toembodiment 12 wherein the C-terminus is an amide of Formula —C(O)—R2 andR2 is —NH₂, —NH-Me, —NH-NHBn, —N(CH₃)—(CH₂)₂-Ph or —NH—(CH₂)₂-Ph; or asalt of the polypeptide.

In embodiment 14, the invention pertains to a polypeptide according toany one of embodiments 1-13 wherein X8 is K; or an amide, an ester or asalt of the polypeptide.

In embodiment 15, the invention pertains to a polypeptide according toany one of embodiments 1 to 14 wherein X9 is G; or an amide, an ester ora salt of the polypeptide.

In embodiment 16, the invention pertains to a polypeptide according toany one of embodiments 1 to 15 wherein X10 is P; or an amide, an esteror a salt of the polypeptide. 19.

In embodiment 17, the invention pertains to a polypeptide according toany one of claims 1 to 16 wherein X11 is Nle or D-Nle; or an amide, anester or a salt of the polypeptide.

In embodiment 17A, the invention pertains to a polypeptide according toanyone of embodiments 1 to 17, wherein the C-terminus consisting of theX11-X12-X13 moiety is selected from Nle-P-phenethylamine, Nle-P—(N-Me)F,Nle-P—NH2, Nle-Phenethylamine, (D-Nle)-phenethylamine, Nle-P-f,D-Nle-a-f, (D-Nle)-NH2, (D-Nle)-f, (D-Nle)-a-d, (D-Nle)-a-y,(D-Nle)-(D-Nva)-f, Nle-P—F, Nle-P-a, Nle-P-Nal and (D-Nle)-(D-Abu)-f, ora salt of the polypeptide. In one particular aspect of this embodiment,the invention relates to a polypeptide according to embodiment 17Awherein the C-terminus consisting of the X11-X12-X13 moiety is selectedfrom (D-Nle)-phenethylamine, (D-Nle)-a-f and f-a-f, or a salt of thepolypeptide.

In one embodiment 17B, the invention pertains to a peptide orpolypeptide of anyone of embodiments 1 to 17, wherein at last two of theamino acids X1, X3, X4 and X7 to X13 are different from thecorresponding amino acids present in Pyr-1-apelin-13. In anotherembodiment, the invention pertains to a peptide or polypeptide of anyoneof embodiments 1 to 18 wherein at least three of the amino acids X1, X3,X4 and X7 to X13 are different from the corresponding amino acidspresent in Pyr-1-apelin-13. In yet another embodiment, the inventionpertains to a peptide or polypeptide of anyone of embodiments 1 to 18wherein at least four of the amino acids X1, X3, X4 and X7 to X13 aredifferent from the corresponding amino acids present in Pyr-1-apelin-13.

In another embodiment, X1, X3, X4 and X7-X13 amino acids are thosedefined by X1, X3, X4, and X7-X13 amino acids in the Examples sectionbelow.

In another embodiment, individual polypeptides according to theinvention are those listed in the Examples section below or apharmaceutically acceptable salt thereof.

Unless specified otherwise, the term “polypeptide of the presentinvention” refers to a polypeptide of Formula (I) and subformulaethereof (Formulae II, III or IV); or an amide, an ester or a saltthereof.

Unless specified otherwise, the terms “polypeptides of the presentinvention,” “peptides of the present invention,” “apelin peptideagonists,” and the like refer to peptides and polypeptides of Formula Iand subformulae thereof (Formulae II, III or IV); or an amide, an esteror a salt thereof. The peptides and polypeptides of the inventiondemonstrate substantially equivalent or improved activity and/or plasmastability over known apelin peptides and polypeptides described herein,including but not limited to wild type apelin, apelin-13 andpyr-1-apelin-13.

The peptides and polypeptides of the invention also encompass peptidesand polypeptides that are at least about 95% identical to the peptidesand polypeptides according to any one of Formulae I, II, III or IV, oran amide, an ester or a salt thereof, as well as to any peptides orpolypeptides specifically listed herein, including but not limited tothe experimental examples.

As used herein, the phrase “homologous amino acid sequence,” orvariations thereof, refers to sequences characterized by a homology, atthe amino acid level, of at least a specified percentage and is usedinterchangeably with “sequence identity.” Homologous amino acidsequences include those amino acid sequences which contain conservativeamino acid substitutions and which polypeptides have the same bindingand/or activity. In some embodiments, an amino acid sequence ishomologous if it has at least 60% or greater, up to 99%, identity with acomparator sequence. In some embodiments, an amino acid sequence ishomologous if it shares one or more, up to 60, amino acid substitutions,additions, or deletions with a comparator sequence. In some embodiments,the homologous amino acid sequences have no more than 5 or no more than3 conservative amino acid substitutions.

Homology may also be at the polypeptide level. The degree or percentageidentity of peptides or polypeptides of the invention, or portionsthereof, and different amino acid sequences is calculated as the numberof exact matches in an alignment of the two sequences divided by thelength of the “invention sequence” or the “foreign sequence”, whicheveris shortest. The result is expressed as percent identity.

A polypeptide comprising an amino acid sequence having a homology ofabout 80-99.9%, preferably 90-99.9% to the amino acid sequence describedin the specific examples, and possessing a plasma stability superior toapelin-13 or pyr-1-apelin-13, fall under the category of the polypeptideof the invention. In one embodiment, the plasma stability improvement isat least 2 fold. In one embodiment, the polypeptide of the invention hasa plasma stability of at least 30 minutes. In another embodiment, thepolypeptide of the invention has a plasma stability of at least 60minutes, or at least 80 minutes, preferably at least 100 min and morepreferably at least 150 minutes.

The term “substantially equivalent” means the nature of thereceptor-binding activity, signal transduction activity and the like isequivalent. Thus, it is allowable that even differences among gradessuch as the strength of receptor binding activity and the molecularweight of the polypeptide are present.

A polypeptide as described herein, or a substantial equivalent thereto,by substitution, deletion, addition or insertion of one or more of aminoacids may be mentioned as polypeptides containing an amino acid sequencesubstantial equivalent(s) in the above sense. A polypeptide as describedherein, or a substantial equivalent thereto, by substitution of 1 to 5,preferably 1 to 3 and more preferably 1 or 2 amino acids with natural orun-natural amino acids may be mentioned as polypeptides containing anamino acid sequence substantial equivalent(s) in the above sense.Further modifications and alterations may include the replacement of anL-amino-acid with a D-amino acid, or other variation including, but notlimited to, phosphorylation, carboxylation, alkylation and the like aslong as the apelin agonistic activity of the peptide or polypeptide offormulae I, II, III or IV is maintained and the plasma stability isimproved over the pyroglutamated form of apelin-13.

In embodiment 19, the invention further pertains to a bioconjugate or amultimer thereof, comprising:

-   -   a. a peptide or polypeptide of Formulae I, II, III or IV, an        amide, salt or ester thereof, according to anyone of the        preceding embodiments;    -   b. a half-life extending moiety;        wherein said peptide or polypeptide and said half-life extending        moiety are covalently linked or fused, optionally via a linker.

In embodiment 19A, the half-life extending moiety is attached to theN-terminus of the peptide of Formula I, II, III or IV, optionally via alinker moiety.

In embodiment 19B, the half-life extending moiety is covalently linkedor fused to the C-terminus of the peptide of Formula I, II, III or IV,optionally via a linker moiety.

In embodiment 19C, the half-life extending moiety is covalently linkedor fused to a side chain of the peptide of Formula I, II, III or IV,e.g. the half-life is covalently linked or fused to an amino group inthe side chain of K, Orn, Dab, Dap, hK or 4-amino-Isn, optionally via alinker moiety. Preferably, the half-life extending moiety is attached tothe N-terminus of the peptide of Formula I, II, III or IV, optionallyvia a linker moiety.

In embodiment 20, the invention pertains to the bioconjugate or amultimer thereof, according to embodiment 19, wherein the half-lifeextending moiety is an IgG constant domain or fragment thereof, a fattyacid or a human Serum Albumin.

In embodiment 21, the invention pertains to a bioconjugate according toembodiments 19 or 20 wherein the half-life extending moiety is a FcLALAmodified Fc fragment with a LALA mutation (L234A, L235A).

In embodiment 22, the invention pertains to the bioconjugate accordingto embodiment 21 wherein the half-life extending moiety is a Fc domainwhich is fused or covalently linked to a polypeptide of Formula I, II,III or IV via a linker and wherein the linker has the following Formula:-[GGGGS]n-, n is 1, 2 or 3 (SEQ ID NO: 7) or the linker is GG or GS andthe polypeptide of Formula I, II, III or IV contains naturally occurringamino acids.

In embodiment 23, the invention pertains to the bioconjugate accordingto embodiment 22 wherein the polypeptide is a polypeptide of Formula Iis selected from QRPC*LSC*KGPMPF (SEQ ID NO: 8), C*RPRLSC*KGPMPF (SEQ IDNO: 9) and QRC*RLSC*KGPMPF (SEQ ID NO: 10), wherein the two amino acidslabeled with “*” represent the amino acids forming a disulfide or amidebond via their side chain.

In embodiment 23A, the invention pertains to a bioconjugate accordingembodiment 22 or 23 wherein the half-life extending moiety is a modifiedFc domain wherein the C-terminal Lysine has been deleted or replacedwith Alanine. Such Fc-variants have been described in co-filedapplication (U.S. provisional application No. 62/015,848: attorneydocket number PAT055781-US-PSP02) and have generated more stable fusionproteins with Apelin peptide/polypeptides.

In embodiment 24, the invention pertains to the bioconjugate or multimerthereof according to embodiment 19 or 20 wherein the half-life extendingmoiety is Human Serum Albumin.

In embodiment 25, the invention pertains to the bioconjugate accordingto embodiment 24 wherein the Human Serum Albumin is chemically linked tothe N-terminus of a polypeptide of any one of Formulae I to IV via alinker of the following Formulae:

wherein x is 1-20, R is linear or branched alkylene, cycloalkyl, aryl ofheteroaryl or combination thereof, R′ is linear or branched alkylene,aryl or cycloalkyl or combination thereof.

In embodiment 26, the invention pertains to the bioconjugate accordingto embodiment 19 or 20 wherein the Human Serum Albumin is chemicallylinked to the C-terminus of a polypeptide of any one of Formulae I to IVvia a linker of the following Formulae:

wherein x is 1-20, R is linear or branched alkylene, cycloalkyl, aryl ofheteroaryl or combination thereof, R′ is linear or branched alkylene,aryl or cycloalkyl or combination thereof.

In other embodiment, the bioconjugate of the invention has the followingformulae:

wherein peptide is the N-terminus of the peptide, A is alanine, H ishistidine, n is 1, 2 or 3, m is 0 or 1, L and L2 are linkers, Cl is amono, di or tricyclic carbocyclic or heterocyclic ring system optionallysubstituted with fluorine and L¹ is a C1-C20 alkylene linker wherein thealkylene chain is optionally substituted with oxo (═O), and wherein oneor more carbon is replaced with O or NH. In a particular aspect of thisembodiment, L and L2 are PEG linkers.

Half-Life Extending Moiety

The half-life extending moiety of the invention can be covalentlyattached, linked, conjugated or fused to a peptide or polypeptideanalog. A half-life extending moiety can be, for example, a polymer,such as polyethylene glycol (PEG), a fatty acid, a cholesterol group, acarbohydrate or olisaccharide; or any natural or synthetic protein,polypeptide or peptide that binds to a salvage receptor. Preferably, thehalf-life extending moiety is covalently linked, optionally via alinker, to plasma protein (albumin and immunoglobulin) with long serumhalf-lives. For example, the half-life extending moiety is an IgGconstant domain or fragment thereof (e.g., the Fc region), Human SerumAlbumin (HSA), or albumin-binding polypeptides or residue (e.g a fattyacid). Preferably, the half-life extending moiety portion of thebioconjugate is human serum albumin, a fatty acid or an Fc region.

Half-life extending moieties include Albumin, which refers to the mostabundant protein in the blood plasma having a molecular weight ofapproximately between 65 and 67 kilodaltons in its monomeric form,depending on species of origin. The term “albumin” is usedinterchangeably with “serum albumin” and is not meant to define thesource of albumin which forms a conjugate with the modified peptides ofthe invention. Thus, the term “albumin” as used herein may refer eitherto albumin purified from a natural source such as blood or serousfluids, or it may refer to chemically synthesized or recombinantlyproduced albumin. Modified peptides or polypeptides of the invention arepreferentially tethered to the free thiol group of the cysteine-34 onthe surface of the albumin, optionally via a linker.

Half-life extending moieties include fatty acids, which can be definedas a C6-70alkyl, a C6-70alkenyl or a C6-70alkynyl chain, each of whichis substituted with at least one carboxylic acid (for example 1, 2, 3 or4 CO2H) and optionally further substituted with hydroxyl group. Examplesof fatty acid are defined by Formulae A1, A2 and A3:

R² is CO₂H, H;R³, R⁴ and R⁵ are independently of each other H, OH, CO₂H, —CH═CH₂ or—C≡CH;Ak¹ is a branched C₆-C₃₀alkylene;q, r and p are independently of each other an integer between 6 and 30;or an amide, an ester or a pharmaceutically acceptable salt thereof.

Examples of fatty acids are selected from:

wherein Ak², Ak³, Ak⁴, Ak⁵ and Ak⁶ are independently a (C₈₋₂₀)alkylene,R⁶ and R⁷ are independently (C₈₋₂₀)alkyl.

More specifically, fatty acids are selected from:

These fatty acid moieties have been described in co-filed application,U.S. provisional application No. 62/015,862 attorney docket numberPAT056274-US-PSP.

Half-life extending moieties include “native Fc” which refers tomolecule or sequence comprising the sequence of a non-antigen-bindingfragment resulting from digestion of whole antibody or produced by othermeans, whether in monomeric or multimeric form, and can contain thehinge region. The original immunoglobulin source of the native Fc ispreferably of human origin and can be any of the immunoglobulins,although IgG1 and IgG2 are preferred. Native Fc molecules are made up ofmonomeric polypeptides that can be linked into dimeric or multimericforms by covalent (i.e., disulfide bonds) and non-covalent association.The number of intermolecular disulfide bonds between monomeric subunitsof native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG,IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). Oneexample of a native Fc is a disulfide-bonded dimer resulting from papaindigestion of an IgG (see Ellison et al., 1982, Nucleic Acids Res. 10:4071-9). The term “native Fc” as used herein is generic to themonomeric, dimeric, and multimeric forms.

Half-life extending moieties include “Fc variant” which refers to amolecule or sequence that is modified from a native Fc but stillcomprises a binding site for the salvage receptor, FcRn (neonatal Fcreceptor). International Publication Nos. WO 97/34631 and WO 96/32478describe exemplary Fc variants, as well as interaction with the salvagereceptor, and are hereby incorporated by reference. Thus, the term “Fcvariant” can comprise a molecule or sequence that is humanized from anon-human native Fc. Furthermore, a native Fc comprises regions that canbe removed because they provide structural features or biologicalactivity that are not required for the bioconjugate of the invention.Thus, the term “Fc variant” comprises a molecule or sequence that lacksone or more native Fc sites or residues, or in which one or more Fcsites or residues has be modified, that affect or are involved in: (1)disulfide bond formation, (2) incompatibility with a selected host cell,(3) N-terminal heterogeneity upon expression in a selected host cell,(4) glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

Half-life extending moieties include Fc variant wherein the C-terminuslysine has been deleted or replaced with alanine.

Half-time extending moieties refer to “Fc domain” which encompassesnative Fc and Fc variants and sequences as defined above. As with Fcvariants and native Fc molecules, the term “Fc domain” includesmolecules in monomeric or multimeric form, whether digested from wholeantibody or produced by other means. In some embodiments of the presentinvention, an Fc domain can be conjugated to a polypeptide of Formula I′or anyone of Formulae I-IV via, for example, a covalent bond between theFc domain and the peptide sequence. Such Fc proteins can form multimersvia the association of the Fc domains and both these Fc proteins andtheir multimers are an aspect of the present invention.

Half-life extending moieties include “modified Fc fragment”, which shallmean an Fc fragment of an antibody comprising a modified sequence. TheFc fragment is a portion of an antibody comprising the CH2, CH3 and partof the hinge region. The modified Fc fragment can be derived from, forexample, IgGI, IgG2, IgG3, or IgG4. FcLALA is a modified Fc fragmentwith a LALA mutation (L234A, L235A), which triggers ADCC with loweredefficiency, and binds and activates human complement weakly. Hessell etal. 2007 Nature 449:101-104. Additional modifications to the Fc fragmentare described in, for example, U.S. Pat. No. 7,217,798.

The term “multimer” as applied to Fc domains or molecule comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently. For example IgG molecules typically form dimersand therefore a bioconjugate comprising a dimeric IgG molecule would befused to two polypeptide chains of Formula I, IA, II, III or IV.

Linker

Any linker group is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer.

The linker is a chemical moiety that contains two reactivegroups/functional groups, one of which can react with the polypeptideand the other with the half-life extending moiety. The two reactivegroups of the linker are linked via a linking group, structure of whichis not critical as long as it does not interfere with the coupling ofthe linker to the peptide and the half-extending moiety.

The linker can be made up of amino acids linked together by peptidebonds. In some embodiments of the present invention, the linker is madeup of from 1 to 20 amino acids linked by peptide bonds, wherein theamino acids are selected from the 20 naturally occurring amino acids. Invarious embodiments, the 1 to 20 amino acids are selected from the aminoacids glycine, serine, alanine, proline, asparagine, glutamine, cysteineand lysine. In some embodiments, a linker is made up of a majority ofamino acids that are sterically unhindered, such as glycine and alanine.In some embodiments, linkers are polyglycines, polyalanines,combinations of glycine and alanine (such as poly(Gly-Ala)), orcombinations of glycine and serine (such as poly(Gly-Ser)). In someembodiments, a linker comprises a majority of amino acids selected fromhistidine, alanine, methionine, glutamine, asparagine and glycine. Insome embodiments, linkers contain poly-histidine moiety. Examples oflinkers are linkers which comprise the motif AH, MHA or AHA. Such motifshave been described in copending applications and co-filed applications,attorney docket numbers U.S. provisional application No. 62/015,862:PAT056274-US-PSP and U.S. provisional application No. 62/015,868:PAT056275-US-PSP, to be beneficial for selective conjugation at theN-terminus of a peptide or polypeptide.

Other examples of linkers comprises the motif GGGGSGGGGSGGGGS (SEQ IDNO: 12), GGGGSGGGGS (SEQ ID NO: 13), GGGGS (SEQ ID NO: 14), GS or GG.

In some other embodiment, the linker comprises recognition motifs forenzyme. An example is the LPXTG/A motif which can be included at theC-terminus wherein X is any amino acid, most commonly an E: Glutamicacid. (L: leucine, P: proline, T: threonine, G: Glycine, A; Alanine).(Carla P. Guimaraes et al.: “Site specific C-terminal and internal looplabeling of proteins using sortase-mediated reactions”, Natureprotocols, vol 8, No 9, 2013, 1787-1799)

In other embodiments, the linker comprises 1 to 20 amino acids which areselected from unnatural amino acids. While a linker of 3-15 amino acidresidues is preferred for conjugation with the half-life extendingmoiety, the present invention contemplates linkers of any length orcomposition. A preferred amino acid linker is O2Oc of the followingformula:

or its repeating units.

The linkers described herein are exemplary, and linkers that are muchlonger and which include other residues are contemplated by the presentinvention. Non-peptide linkers are also contemplated by the presentinvention.

The linking portion of the linker may comprise one or more alkyl groups,alkoxy groups, alkenyl groups, cycloalkyl groups, aryl groups,heteroaryl groups and heterocyclic groups or combination thereof. Forexample, alkyl linkers such as such as —NH—(CH₂)z-C(O)— or—S—(CH₂)z-C(O)— or —O—(CH₂)z-C(O)— wherein z is 2-20 can be used. Thesealkyl linkers can further be substituted by any non-sterically hinderinggroup, including, but not limited to, a lower alkyl (e.g., C1-C6), loweracyl, halogen (e.g., Cl, Br), CN, NH2, or phenyl.

The linker can also be of polymeric nature. The linker may includepolymer chains or units that are biostable or biodegradable. Polymerswith repeat linkage may have varying degrees of stability underphysiological conditions depending on bond lability. Polymers maycontain bonds such as polycarbonates (—O—C(O)—O—), polyesters(—C(O)—O—), polyurethanes (—NH—C(O)—O—), polyamide (—C(O)—NH—). Thesebonds are provided by way of examples, and are not intended to limit thetype of bonds employable in the polymer chains or linkers of theinvention. Suitable polymers include, for example, polyethylene glycol(PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids,divinylether maleic anhydride, N-(2-hydroxypropyl)-methacrylicamide,dextran, dextran derivatives, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, starch and starch derivatives, polyalkyleneglycol and derivatives thereof, copolymers of polyalkylene glycols andderivatives thereof, polyvinyl ethyl ether, and the like and mixturesthereof. A polymer linker is for example PEG. An exemplary non-peptidelinker is a polyethylene glycol linker:

wherein y is so that the linker has a molecular weight of 100 to 5000kD, for example, 100 to 500 kD.

Preferably, the linking moiety contains one or more amino acid moietiessuch as for example (O2Oc) unit or Glycine or serine,C₁₋₄alkylene-C(O)—, C₁₋₄alkylene, —NH—C₂₋₆alkylene-NH— or—NH—CH₂CH₂—O—CH₂CH₂—NH— diamino units or combination thereof and thelinking moiety linked 2 reactive groups or functional groups.

Preferably, the reactive groups or functional groups are maleimide,thiol or pyridine-2-yldisulfanyl.

Preparation of Peptide or Polypeptide and Peptide-Linker Construct forAttachment to a Half-Life Extending Moiety:

The apelin peptides and polypeptides and/or peptide-linker construct ofthe invention may be produced by either synthetic chemical processes orby recombinant methods or combination of both methods. The Apelinpeptides and/or peptide-linker constructs may be prepared as full-lengthor may be synthesized as non-full length fragments and joined. Thepeptides and polypeptides of the present invention can be produced bythe per se known procedures for peptide synthesis. The methods forpeptide synthesis may be any of a solid-phase synthesis and aliquid-phase synthesis. Thus, the peptide and polypeptide of interestcan be produced by condensing a partial peptide or amino acid capable ofconstituting the protein with the residual part thereof and, when theproduct has a protective group, the protective group is detachedwhereupon a desired peptide can be manufactured. The known methods forcondensation and deprotection include the procedures described in thefollowing literature (1)-(5).

-   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, 1966,-   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,    1965,-   (3) Nobuo Izumiya et al. Fundamentals and Experiments in Peptide    Synthesis, Maruzen, 1975,-   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment    Series 1, Protein Chemistry IV, 205, 1977, and-   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,    Peptide Synthesis, Hirokawa Shoten.

After the reaction, the peptide can be purified and isolated by acombination of conventional purification techniques such as solventextraction, column chromatography, liquid chromatography, andrecrystallization. Where the peptide isolated as above is a freecompound, it can be converted to a suitable salt by the known method.Conversely where the isolated product is a salt, it can be converted tothe free peptide by the known method.

The amide of polypeptide can be obtained by using a resin for peptidesynthesis which is suited for amidation. The resin includes chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenz-hydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, 2-chlorotritylchloride resin, and so on. Using such a resin, amino acids whose α-aminogroups and functional groups of side-chain have been suitably protectedare condensed on the resin according to the sequence of the objectivepeptide by various condensation techniques which are known per se. Atthe end of the series of reactions, the peptide or the protected peptideis removed from the resin and the protective groups are removed and ifnecessary, disulfide bonds are formed to obtain the objectivepolypeptide.

For the condensation of the above-mentioned protected amino acids, avariety of activating reagents for peptide synthesis can be used such asHATU, HCTU or e.g. a carbodiimide. The carbodiimide includes DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation with sucha reagent, a racemization inhibitor additive, e.g. HOBt or Oxyma Purecan be used. The protected amino acid can be directly added to the resinalong with the activation reagents and racemization inhibitor or bepre-activated as symmetric acid anhydride, HOBt ester, or HOOBt esterthen added to the resin. The solvent for the activation of protectedamino acids or condensation with the resin can be properly selected fromamong those solvents which are known to be useful for peptidecondensation reactions. For example, N,N-dimethylformamide,N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide,DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran,acetonitrile, ethyl acetate, or suitable mixtures of them can bementioned.

The reaction temperature can be selected from the range hitherto-knownto be useful for peptide bond formation and is usually selected from therange of about −20° C.-50° C. The activated amino acid derivative isgenerally used in a proportion of 1.5-4 fold excess. If the condensationis found to be insufficient by a test utilizing the ninhydrin reaction,the condensation reaction can be repeated to achieve a sufficientcondensation without removing the protective group. If repeatedcondensation still fails to provide a sufficient degree of condensation,the unreacted amino group can be acetylated with acetic anhydride oracetylimidazole.

The protecting group of amino group for the starting material amino acidincludes Z, Boc, tertiary-amyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, or Fmoc. The carboxy-protecting group that canbe used includes but is not limited to the above-mentioned C₁₋₆ alkyl,C₃₋₈ cycloalkyl and C₆₋₁₀aryl-C₁₋₂alkyl as well as 2-adamantyl,4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl,benzyloxycarbonylhydrazido, tertiary-butoxycarbonylhydrazido, andtritylhydrazido.

The hydroxy group of serine and threonine can be protected byesterification or etherification. The group suited for saidesterification includes carbon-derived groups such as lower alkanoylgroups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc.,benzyloxycarbonyl, and ethoxycarbonyl. The group suited for saidetherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.The protective group for the phenolic hydroxyl group of tyrosineincludes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, and tertiary-butyl.

The protecting group of imidazole for histidine includes Tos,4-methoxy-2,3,6-tri ethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes thecorresponding acid anhydride, azide and active esters, e.g. esters withalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc. The activatedamino group of the starting amino acid includes the correspondingphosphoramide.

The method for elimination of protective groups includes catalyticreduction using hydrogen gas in the presence of a catalyst such aspalladium black or palladium-on-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid, or a mixture of such acids, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, reductionwith sodium metal in liquid ammonia. The elimination reaction by theabove-mentioned acid treatment is generally carried out at a temperatureof −20° C.-40° C. and can be conducted advantageously with addition of acation acceptor such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. The2,4-dinitrophenyl group used for protecting the imidazole group ofhistidine can be eliminated by treatment with thiophenol, while theformyl group used for protecting the indole group of tryptophan can beeliminated by alkali treatment with dilute sodium hydroxide solution ordilute aqueous ammonia as well as the above-mentioned acid treatment inthe presence of 1,2-ethanedithiol, 1,4-butanedithiol.

The method for protecting functional groups which should not take partin the reaction of the starting material, the protective groups that canbe used, the method of removing the protective groups, and the method ofactivating the functional groups that are to take part in the reactioncan all be selected judicially from among the known groups and methods.

An another method for obtaining the amide form of the polypeptidecomprises amidating the α-carboxyl group of the C-terminal amino acid atfirst, then extending the peptide chain to the N-side until the desiredchain length, and then selectively deprotecting the α-amino group of theC-terminal peptide and the α-carboxy group of the amino acid or peptidethat is to form the remainder of the objective polypeptide andcondensing the two fragments whose α-amino group and side-chainfunctional groups have been protected with suitable protective groupsmentioned above in a mixed solvent such as that mentioned hereinbefore.The parameters of this condensation reaction can be the same asdescribed hereinbefore. From the protected peptide obtained bycondensation, all the protective groups are removed by theabove-described method to thereby provide the desired crude peptide.This crude peptide can be purified by known purification procedures andthe main fraction be lyophilized to provide the objective amidatedpolypeptide. To obtain an ester of the polypeptide, the a-carboxyl groupof the C-terminal amino acid is condensed with a desired alcohol to givean amino acid ester and then, the procedure described above forproduction of the amide is followed.

Alternatively, recombinant expression methods are particularly useful.Recombinant protein expression using a host cell (a cell artificiallyengineered to comprise nucleic acids encoding the sequence of thepeptide and which will transcribe and translate, and optionally, secretethe peptide into the cell growth medium) is used routinely in the art.For recombinant production process, a nucleic acid coding for amino acidsequence of the peptide would typically be synthesized by conventionallymethods and integrated into an expression vector. Such methods isparticularly preferred for manufacture of the polypeptide compositionscomprising the peptides fused to additional peptide sequences or otherproteins or protein fragments or domains. The host cell can optionallybe at least one selected from E. Coli, COS-1, COS-7, HEK293, BHT21, CHO,BSC-1, Hep G2, 653, SP2/0, 293, heLa, myeloma, lymphoma, yeast, insector plant cells, or any derivative, immortalized or transformed cellthereof.

The modified therapeutic peptides or polypeptides and/or peptide-linkerconstruct include reactive groups which can react with availablereactive functionalities on the half-life extending moiety to form acovalent bond. Reactive groups are chemical groups capable of forming acovalent bond. Reactive groups can generally be carboxy, phosphoryl,acyl group, ester or mixed anhydride, maleimide, imidate,pyridine-2-yl-disulfanyl, thereby capable of forming a covalent bondwith functionalities like amino group, hydroxyl group, carboxy group ora thiol group at the target site of the Albumin or Fc domain. Reactivegroups of particular interest for linking to an Albumin includemaleimido-containing groups and pyridine-2-yl-disulfanyl containinggroup.

Functionalities are groups on Albumin or Fc domain to which reactivegroups on modified peptides or polypeptides are capable of reacting withto form covalent bonds. Functionalities include hydroxyl groups forbonding with ester reactive entities, thiol groups for reacting withmaleimides, maleimido-containing groups or pyridine-2-yldisulfanyl,imidates and thioester groups; amino groups for bonding to carboxylicacid, phosphoryl groups, acyl groups; and carboxy groups for reactingwith hydrazine, hydrazide or hydroxylamine.

Schemes 1 to 3 describe the synthesis of peptide-Linker constructwherein the peptide is a peptide according to anyone of Formulae I toIV.Scheme 1 describes the synthesis of a maleimide containing linkerattached to the N-terminus of a polypeptide of Formula I to IV.

The N-terminus of the peptide is coupled with one or more O2Oc aminoacid units (x is 1 to 20, preferably 1 to 10 and more preferably 3 to 6)according to well established amide coupling chemistry to generate (1A).The terminal amino functionality of (1A) is reacted with an activatedacid (1B) wherein R is linear or branched alkylene, aryl, heteroaryl,cycloalkyl or combination thereof, in order to generate thepeptide-maleimide containing linker construct (1C). The activated acid(1B) is commercially available or readily available from itscorresponding carboxylic acid according to technique known to someone ofordinary skill in the art. Preferably, R is a linear alkylene, and morepreferably R is —CH₂—CH₂—. Alternatively, for peptides containing anamino functionality in the side chain (for example peptide containing alysine), orthogonal protecting group such as Alloc is required prior tothe coupling reaction, followed by additional deprotection step in orderto obtain (1C).Scheme 2A and 2B describe the synthesis of pyridine-2-yl-disulfanylcontaining linker attached to the N-terminus of a polypeptide accordingto any one of Formula I to IV.

Peptide-Linker construct (1A) is prepared as described in Scheme 1 andis further reacted with an activated acid of Formula (2A) wherein R′ isa linear or branched alkylene, to generate apeptide-pyridine-2-yl-disulfanyl containing linker construct (2B).Activated acid (2A) is commercially available or is readily availablefrom its corresponding carboxylic acid according to techniques known tosomeone of ordinary skill in the art. Preferably R′ is —CH₂—CH₂—.Alternatively, Peptide-linker construct (2C) can be prepared usingHO₂C—R′—SH, or a protected form thereof (e.g. trityl or Acm groups,requiring additional deprotection steps), and further reacted with (2D)to generate peptide-pyridine-2-yl-disulfanyl containing linker construct(2B).Similar reactive groups are attached to the C-terminus of the peptide ina similar way as described in Schemes 1, 2A and 2B using a diamino unitsuch as for example —NH—CH₂CH₂—NH— or —NH—CH₂CH₂—O—CH₂CH₂—NH—. Nonlimiting examples of such peptide-linker conducts are:

Alternatively maleimide or pyridine-2-yl-disulfanyl reactive group canbe attached to a polypeptide according to any one of Formula I to IVaccording to scheme 3A, 3B and 3C:

The carboxylic acid group at the C-terminus of the peptide is coupledwith one or more O2Oc amino acid units using standard amide couplingconditions to generate (3A). The terminal carboxylic acid functionalityreacts with the amino group of (3B) or (3C) wherein R and R′ are asdefined above, in order to generate the activated Peptide-LinkerConstructs (3D) or (3E). Additionally, when a peptide contains a carboxyfunctionality side chain (e.g. Glu or Asp), orthogonal protecting group(e.g. O-Allyl) and additional deprotection steps are required.

Peptide-Linker Construct 3F can be obtained using a cysteamine2-chlorotrityl Resin and then reacted with 3G or 3H to generatepeptide-linker construct 3I or 3E respectively.

Peptide-Linker Construct (3J) can be obtained from a diamine resin andbe further reacted with (1B) or (2A) to generate a Peptide-LinkerConstruct of Formula (3K) or (3L) respectively.Schemes 1 to 3C describe Peptide-Linker Constructs, more particularlyfor use in the preparation of a bioconjugate with Albumin. The maleimidereactive group and the pyridine-2-yl-disulfanyl reactive group reactswith the —SH functionality of Cysteine 34 of the albumin.Schemes 3D and 3E describes preparation of peptide-linker constructs foruse in an azide-alkyne Huisgen cycloaddition, more commonly known asclick chemistry.

wherein m is 0 or 1, Cl is a mono, di or tricyclic carbocyclic orheterocyclic ring system optionally substituted with fluorine, L¹ is aC1-C20 alkylene linker wherein the alkylene chain is optionallysubstituted with oxo (═O), and wherein one or more carbon is replacedwith O or NH. Cycloalkyne moieties (3 Da) are readily available fromcommercial sources. Additionally, cyclic alkyne in click chemistry forprotein labeling has been described in US 2009/0068738 which is hereinincorporated by reference. Specific examples have been described below(example 20). The click handle can be introduced at the N-terminus ofthe peptide or on a lysine residue side chain.

Scheme 3E describes the introduction of an Azido lysine residue at theN-terminus of an apelin peptide optionally via a linker L (such as forexample one or more amino acids selected from Glycine and serine). Theazide functionality acts as a handle for click chemistry. Specificexamples have been described in co-filed application (attorney docketnumber PAT055418-US-PSP2).

Preparation of Half-Life Extending Moiety-Linker Construct:

Scheme 3F and 3G describes the preparation of a fatty acid-linkerconstruct.

wherein FA is fatty acid, L2 is a linking moiety (for example PEG), NHSis N-hydroxysuccinimide. Such fatty acid-linker constructs are used forconjugation using click chemistry. In instances wherein the fatty acidcontains functionalities such as hydroxyl or additional carboxylic acid,protection of such functionalities may be required.

Wherein FA, NHS and L2 are defined above in Scheme 3F. Such fatty acidconstructs are used for conjugation with an amino functionality on thepeptide, preferably the N-terminus.Scheme 3H describes the preparation of a Fc-linker construct

AH-Fc is a construct containing the sequence AH- at the N-terminus ofthe Fc. The construct is prepared using recombinant methods. TheAH-sequence allows for selective modification of the N-terminus at a lowpH. Such selective modification has been disclosed in cofiledapplications (U.S. provisional application No. 62/015,868: attorneydocket number PAT056275-US-PSP and U.S. provisional application No.62/015,862: PAT056274-US-PSP). Click handle is therefore introduced atthe N-terminus of the Fc construct.

In yet another embodiment, the Fc construct is modified at theC-terminus to introduce a small Sortase recognition motif (LPXTG/A).

Such Fc-recognition motif is prepared using recombinant methods. Exampleof such construct is: Fc-[GGGG]n-LPETGGLEVLFQGP (SEQ ID NO: 15) whereinthe GGLEVLFQGP (SEQ ID NO: 16) is clipped during sortase treatment.

Preparation of the Fc APJ Peptide Fusion Protein

The biologically generated multimerized molecule, such as an antibody Fccomprising at least a part of cysteine containing region known as thehinge can be prepared from recombinant expressed protein product whichhas been secreted in multimerized (e.g. dimeric) form. The presentinvention also include modified Fc fusion proteins wherein the aminoacid sequence of the Fc region has been altered relative to the aminoacid sequence of the Fc- or constant region found in a naturallyoccurring antibody. For example, Fc-fusion protein may be engineered(i.e. modified) with mutations in order to obtain desiredcharacteristics of FcRn binding affinity/or serum half-life. Example ofmodified Fc-fusion proteins have been disclosed in U.S. Pat. No.7,217,798, which is incorporated by reference.

Fc-fusion proteins of this invention may also be altered synthetically,e.g. by attachment of the linker moiety and the peptide or polypeptidemoiety. In addition, “modified” Fc-fusion proteins with Fc domainderived from recombinant antibodies can be made in any expressionsystems including both prokaryotic and eukaryotic expression system orusing phage display methods.

Fc-Linker Constructs such as Fc-[GGGGS] (SEQ ID NO: 14), Fc-[GGGGS]2(SEQ ID NO: 13), Fc-[GGGGS]3 (SEQ ID NO: 12), Fc-GG and Fc-GS, aredescribed below in the experimental part. The [GGGGS] (SEQ ID NO: 14),[GGGGS]2 (SEQ ID NO: 13), [GGGGS]3 (SEQ ID NO: 12), GS and GG linker areattached either to the C-terminus of the Fc domain or to the N-terminusof the Fc domain, wherein Fc is a native Fc or a variant thereof.Example of Fc variant includes a Fc wherein the C-terminal Lysine hasbeen deleted or replaced with Alanine. Such Fc variant has beendescribed in co-filed application (attorney docket NoPAT055781-US-PSP02)

Bioconjugates

In one embodiment of the present invention, a peptide or polypeptideaccording to anyone of Formula I to IV is conjugated(chemically/covalently attached) to the thiol functionality of cysteine34 of the albumin. In one aspect of this embodiment, the Albumin-Peptiderefers to a bioconjugate in which the Albumin is conjugated (chemicallylinked) to the N-terminus of the peptide. In yet another embodiment, theAlbumin-Peptide refers to a bioconjugate in which the Albumin isconjugated (chemically linked) to the C-terminus of the peptide.

In another embodiment of the present invention, a peptide or polypeptideaccording to anyone of Formula I to IV is fused to one or more domainsof an Fc region of human IgG. Antibodies comprise two functionallyindependent parts, a variable domain known as “Fab,” that binds anantigen, and a constant domain known as “Fc,” that is involved ineffector functions such as complement activation and attack byphagocytic cells. An Fc has a long serum half-life, whereas a Fab isshort-lived (Capon et al., 1989, Nature 337: 525-31). when joinedtogether (with a therapeutic peptide or polypeptide, an Fc domain canprovide longer half-life (C. Huang, Curr. Opin. Biotechnol., 2009, 20,692-699).

In one embodiment, the Fc-Peptide refers to a bioconjugate in which theFc sequence is fused to the N-terminus of the peptide according toanyone of Formulae I to IV. In another embodiment, Peptide-Fc refers toa bioconjugate in which the Fc sequence is fused to the C-terminus ofthe peptide.

The Fc region can be a naturally occurring Fc region, or can be alteredto improve certain qualities, such as therapeutic qualities, circulationtime, or reduced aggregation.

Useful modifications of protein therapeutic agents by fusion with the“Fc” domain of an antibody are discussed in detail in PCT PublicationNo. WO 00/024782. This document discusses linkage to a “vehicle” such aspolyethylene glycol (PEG), dextran, or an Fc region.

Preferred embodiments of the invention are bioconjugate comprising apeptide or polypeptide according to anyone of preceding embodiments anda half life extending moiety, wherein the half-life extending moiety isa Fc domain fused to a polypeptide of Formula I, III, IV or V via alinker. In one aspect of this invention, the linker has the followingFormula: -[GGGGS]n-, n is 1, 2 or 3 (SEQ ID NO: 7) or the linker is GGor GS, and the polypeptide of Formula I, II, III or IV containsnaturally occurring amino acids. Examples of polypeptides of Formula I,II, III or IV suitable for fusion with the Fc domain are:QRPC*LSC*KGPMPF (SEQ ID NO: 8), C*RPRLSC*KGPMPF (SEQ ID NO: 9) andQRC*RLSC*KGPMPF (SEQ ID NO: 10). One preferred aspect of thisembodiments are Fc-Peptide fused bioconjugate as defined above,comprising a modified Fc fragment (e.g., an FcLALA) and a peptide orpolypeptide of anyone of Formulae I to IV, as defined herein.

In yet another embodiment, the invention pertains to a bioconjugateaccording to any one of the preceding embodiments wherein the half-lifeextending moiety is a modified Fc domain wherein the C-terminal Lysinehas been deleted or replaced with Alanine.

Peptides fused to an Fc region have been found to exhibit asubstantially greater half-life in vivo than the unfused counterpart.Also, a fusion to an Fc region allows for dimerization/multimerizationof the polypeptide.

In another embodiments of the invention are bioconjugate comprising apeptide or polypeptide according to anyone of Formulae I-IV and a halflife extending moiety, wherein the half-life extending moiety is a Fcdomain which is chemically linked to a polypeptide.

Preparing Conjugates:

Schemes 4 and 5 illustrate chemical reactions for conjugation of an APJagonist peptide or a peptide according to anyone of Formula I to IV anda half-life extending moiety such as an Fc domain or albumin.Scheme 4 illustrates the conjugation of a peptide-linker of Formula 4Awith Cysteine 34 of Albumin

wherein L represent a linking moiety between the peptide and themaleimide functionality. In a particular embodiment, L is a linkingmoiety as disclosed in Scheme 1, 3A, 3B or 3C.Scheme 5 illustrates the conjugation of a peptide-linker construct ofFormula 8A with Cysteine 34 of Albumin.

wherein L represents a linking moiety between the peptide and the—S—S-Pyridine functionality. In a particular embodiment, L is a linkingmoiety as disclosed in schemes 2, 3A, 3B or 3C.

Other method of conjugation have been described in copending andco-filed application (U.S. provisional application No. 62/015,862:attorney docket numbers PAT056274-US-PSP, U.S. provisional applicationNo. 62/015,868:PAT056275-US-PSP and U.S. provisional application No.62/015,848 PAT055781-US-PSP02). Such method includes selectiveN-acylation of a peptide and is summarized in Scheme 6.

wherein AH- is a linker introduced on N-terminus of the peptide tofacilitate reaction at the N-terminus, H is histidine, A is Alanine, FAis a fatty acid as described supra, for example a fatty acid of FormulaA1 to A3, and L is a linking moieties (for example a PEG linkingmoiety). The Fatty acid Linker construct 6a (prepared as shown in Scheme3G) is selectively introduced onto the peptide at the N-terminus whenusing low pH condition. Such method has been described in cofiled patentapplications (U.S. provisional application No. 62/015,868: attorneydocket number PAT056275-US-PSP and U.S. provisional application No.62/015,848 PAT055781-US-PSP02).Schemes 7 and 8 describes formation of conjugates according to theinstant invention using click chemistry.

Methods for making conjugates and peptide-linker constructs as describedin Schemes 1-5 have also been described and exemplified in co-filed USapplications (U.S. provisional application No. 61/858,251: Attorneydockets numbers: PAT055326-US-PSP3 and U.S. provisional application No.61/858,303: PAT055781-US-PSP) which are hereby incorporated byreference.

Scheme 9 describes the conjugation of an APJ peptide with a Fc constructusing a sortase enzyme

(SEQ ID NOS 17 and 11, respectively, in order of appearance)

wherein n is 1, 2 or 3, L is an optional linker (for example apolyglycine linker)

Pharmaceutical Compositions

The polypeptides of the instant invention, or an amide, an ester of asalt thereof, or a bioconjugate thereof, may be administered in any of avariety of ways, including subcutaneously, intramuscularly,intravenously, intraperitoneally, intranasally, inhalationally, orallyetc. Particularly preferred embodiments of the invention employcontinuous intravenous administration of the polypeptides of the instantinvention, or an amide, ester, or salt thereof or a bioconjugatethereof. The polypeptides or bioconjugates of the instant invention maybe administered as a bolus or as a continuous infusion over a period oftime. An implantable pump may be used. In certain embodiments of theinvention, intermittent or continuous polypeptides or bioconjugatesadministration is continued for one to several days (e.g., 2-3 or moredays), or for longer periods of time, e.g., weeks, months, or years. Insome embodiments, intermittent or continuous polypeptide administrationis provided for at least about 3 days. In other embodiments,intermittent or continuous polypeptide or bioconjugate administration isprovided for at least about one week. In other embodiments, intermittentor continuous polypeptide or bioconjugate administration is provided forat least about two weeks. It may be desirable to maintain an averageplasma polypeptide concentration above a particular threshold valueeither during administration or between administration of multipledoses. A desirable concentration may be determined, for example, basedon the subject's physiological condition, disease severity, etc. Suchdesirable value(s) can be identified by performing standard clinicaltrials. Alternatively, the peptides and conjugates thereof could bedelivered orally via FcRn mechanism. (Nat Rev Immunol. 7(9), 715-25,2007; Nat Commun. 3; 3:610, 2012, Am J Physiol Gastrointest LiverPhysiol 304: G262-G270, 2013).

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a polypeptide of the present invention or andamide, an ester or a salt thereof or a bioconjugate thereof, and one ormore pharmaceutically acceptable carriers. The pharmaceuticalcomposition can be formulated for particular routes of administrationsuch as oral administration, parenteral administration, and rectaladministration, etc. In addition, the pharmaceutical compositions of thepresent invention can be made up in a solid form (including withoutlimitation capsules, tablets, pills, granules, powders orsuppositories), or in a liquid form (including without limitationsolutions, suspensions or emulsions). The pharmaceutical compositionscan be subjected to conventional pharmaceutical operations such assterilization and/or can contain conventional inert diluents,lubricating agents, or buffering agents, as well as adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifers and buffers, etc.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. Preferred pharmaceutical formulations are stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Ingeneral, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Certain injectable compositions are aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. Said compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1-75%, or contain about 1-50%, of theactive ingredient.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive therapeutic agents arepreferably delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. It is noted that the lungsprovide a large surface area for systemic delivery of therapeuticagents.

The agents may be encapsulated, e.g., in polymeric microparticles suchas those described in U.S. publication 20040096403, or in associationwith any of a wide variety of other drug delivery vehicles that areknown in the art. In other embodiments of the invention the agents aredelivered in association with a charged lipid as described, for example,in U.S. publication 20040062718. It is noted that the latter system hasbeen used for administration of a therapeutic polypeptide, insulin,demonstrating the utility of this system for administration of peptideagents.

Systemic administration can also be by transmucosal or transdermalmeans.

Suitable compositions for transdermal application include an effectiveamount of a polypeptide of the invention with a suitable carrier.Carriers suitable for transdermal delivery include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound of the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Suitable compositions for topical application, e.g., to the skin andeyes, include aqueous solutions, suspensions, ointments, creams, gels orsprayable formulations, e.g., for delivery by aerosol or the like. Suchtopical delivery systems will in particular be appropriate for dermalapplication. They are thus particularly suited for use in topical,including cosmetic, formulations well-known in the art. Such may containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

As used herein a topical application may also pertain to an inhalationor to an intranasal application. They may be conveniently delivered inthe form of a dry powder (either alone, as a mixture, for example a dryblend with lactose, or a mixed component particle, for example withphospholipids) from a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray, atomizer ornebuliser, with or without the use of a suitable propellant.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, etc.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of thepolypeptides of this invention and, which typically are not biologicallyor otherwise undesirable. In many cases, the polypeptides of the presentinvention are capable of forming acid and/or base salts by virtue of thepresence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulfornate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,sulfosalicylic acid, and the like. Pharmaceutically acceptable baseaddition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, chlorinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound, a basic or acidic moiety, byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, use of non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile isdesirable, where practicable. Lists of additional suitable salts can befound, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., MackPublishing Company, Easton, Pa., (1985); and in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

Method of the Invention:

Apelin family of peptides is the only known natural family of ligandsfor the G protein coupled APJ receptor. Apelin gene encodes a 77 aminoacid polypeptide, which gets processed into biologically active forms ofapelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13,apelin-12 and pyroglutamate modified form of apelin-13 (Pyr¹-apelin-13).Any one of these apelin peptides, upon binding to APJ receptor,transduces the signal via Gi and Gq proteins. In cardiomyocytes, Gi orGq coupling leads to changes in intracellular pH, PLC activation, andIP3 production that enhance myofilament calcium sensitivity andultimately result in increased cardiac contractility. Gi couplinginhibits activated Gs, adenylyl cyclase and cAMP production andincreases pAkt levels leading to cardioprotection. In vascularendothelial cells, APJ activation via Gi, pAKT leads to increased nitricoxide (NO) production, which increases smooth muscle relaxationresulting in overall vasodilation.

Patients with chronic stable heart failure have occasional acuteepisodes of decompensation, where cardiac contractility declines furtherand symptoms worsen. These exacerbations are referred to as acutedecompensated heart failure (ADHF). Current therapies for ADHF includediuretics, vasodilators, and inotropes, which directly increase cardiaccontractility. Current intravenous inotropes (dobutamine, dopamine,milrinone, levosimendan) are well known for their adverse events such asarrhythmia and increased long-term mortality. The synthetic apelinpolypeptide analogs of the instant invention, or bioconjugate thereof,provide a therapy for ADHF that increases cardiac contractility withoutarrhythmogenic or mortality liabilities and address the enormous unmetmedical need in chronic heart failure.

Indeed, acute apelin treatment (5 min) in humans results in coronaryvasodilatation and improved cardiac output. However, native apelinsexhibit a very short t_(1/2) (seconds) and duration of action (fewminutes) in vivo. The potent synthetic apelin peptide agonists of theinstant invention, or bioconjugate thereof, have longer half livescompared to the native apelin.

Activation of APJ receptor in cardiomyocytes a) improve cardiaccontractility via Gi/Gq, PLC and Ca2+, and b) provide cardioprotectionvia Gi, pAkt activation, but without increasing cAMP (as seen with otherinotropes). In addition, APJ agonism in endothelial cells leads toarterial vasodilation, which further benefits heart failure by unloadingthe work of left ventricle. Taken together the synthetic apelinpolypeptide analogs can improve overall cardiac function, reducearrhythmogenesis and provide survival benefit.

More recently, there have been a number of preclinical researchpublications focusing on the potential involvement of Apelin in diabetesand insulin resistance. Apelin has been shown to 1) lower blood glucoselevels by improving glucose uptake in muscle, adipose and heart, 2)protect pancreatic beta cells from ER stress and subsequent apoptosis,3) lower the insulin secretion in beta cells, and 4) regulatecatecholamine induced lypolysis in adipose tissue. Activation of pAKTpathway has been implicated in these processes.

The polypeptides according to anyone of formulae I to IV, or apharmaceutically acceptable salt thereof, in free form or inpharmaceutically acceptable salt form, or a bioconjugate thereof,exhibit valuable pharmacological properties, e.g. APJ receptor agonismproperties, e.g. as indicated in in vitro and in vivo tests as providedin the next sections and are therefore indicated for therapy.

Polypeptides of the invention or a pharmaceutically acceptable saltthereof, or bioconjugates thereof, may be useful in the treatment of anindication selected from acute decompensated heart failure (ADHF),chronic heart failure, pulmonary hypertension, atrial fibrillation,Brugada syndrome, ventricular tachycardia, atherosclerosis,hypertension, restenosis, ischemic cardiovascular diseases,cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes(including gestational diabetes), obesity, peripheral arterial disease,cerebrovascular accidents, transient ischemic attacks, traumatic braininjuries, amyotrophic lateral sclerosis, burn injuries (includingsunburn) and preeclampsia.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof, or a bioconjugate thereof, for the treatment of a diseasewhich is associated with the APJ receptor activity. In a furtherembodiment, the therapy is selected from a disease which is responsiveto the agonism of the APJ receptor. In another embodiment, the diseaseis selected from the afore-mentioned list, suitably acute decompensatedheart failure. In yet another subset of this embodiment, the presentinvention provides the use of a polypeptide of anyone of formulae I toIV, or an amide, ester or a salt thereof, or a bioconjugate thereof, inthe manufacture of a medicament, for the treatment of a disease which isassociated with the APJ receptor activity.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof, or a bioconjugate thereof, in therapy. In a furtherembodiment, the therapy is selected from a disease which may be treatedby activation (agonism) of the APJ receptor.

In another embodiment, the invention provides a method of treating adisease which is responsive to the agonism of the APJ receptor,comprising administration of a therapeutically acceptable amount of apolypeptide of anyone of formulae I to IV, or an amide, an ester of asalt thereof, or a bioconjugate thereof. In a further embodiment, thedisease is selected from the afore-mentioned list, suitably acutedecompensated heart failure.

In yet another subset of this embodiment, the invention provides amethod of treating a disease which is associated with the activity ofthe APJ receptor comprising administration of a therapeuticallyacceptable amount of a polypeptide of anyone of formulae I to IV, or anamide, an ester or a salt thereof or a bioconjugate thereof.

The effective amount of a pharmaceutical composition or combination ofthe invention to be employed therapeutically will depend, for example,upon the therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the fusion protein variant is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the dual function protein in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

The term “a therapeutically effective amount” of a polypeptide of thepresent invention or a bioconjugate thereof, refers to an amount of thepolypeptide of the present invention (or bioconjugate) that will elicitthe biological or medical response of a subject, for example,amelioration of a symptom, alleviation of a condition, slow or delaydisease progression, or prevention of a disease, etc. In onenon-limiting embodiment, the term “a therapeutically effective amount”refers to the amount of the polypeptide of the present invention that,when administered to a subject, is effective to (1) at least partiallyalleviate, inhibit, prevent and/or ameliorate a condition, a disorder ora disease or a symptom thereof (i) ameliorated by the activation of theAPJ receptor or (ii) associated with the activity of the APJ receptor,or (iii) characterized by abnormal activity of the APJ receptor; or (2)activate the APJ receptor.

In another non-limiting embodiment, the term “a therapeuticallyeffective amount” refers to the amount of the polypeptide of the presentinvention that, when administered to a cell, or a tissue, or anon-cellular biological material, or a medium, is effective to at leastpartially activate the APJ receptor. As will be appreciated by those ofordinary skill in the art, the absolute amount of a particular agentthat is effective may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the target tissue, etc.Those of ordinary skill in the art understand that “a therapeuticallyeffective amount” may be administered in a single dose or may beachieved by administration of multiple doses. For example, in the caseof an agent to treat heartfailure, an effective amount may be an amountsufficient to result in clinical improvement of the patient, e.g.,increased exercise tolerance/capacity, increased blood pressure,decrease fluid retention, and/or improved results on a quantitative testof cardiac functioning, e.g., ejection fraction, exercise capacity (timeto exhaustion), etc.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, fish, birds and the like. In certain embodiments, the subject is aprimate. In yet other embodiments, the subject is a human.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence, onset, or development of one ormore symptoms of a disorder in a subject resulting from theadministration of a therapy (e.g., a therapeutic agent), or theadministration of a combination of therapies (e.g., a combination oftherapeutic agents).

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The activity of a polypeptide according to the present invention can beassessed by the following in vitro methods described below.

hAPJ Calcium Flux Assay:

Chem-5 APJ stable cells (Millipore #HTS068C) were plated in 384-wellformat with 10,000 cells/well in 25 ul growth media, then grown 24 hoursin a 37° C. tissue culture incubator. One hour before the assay, 25ul/well FLIPR Calcium 4 dye (Molecular Devices R8142) with 2.5 mMprobenecid was added, and cells were incubated one hour in a 37° C.tissue culture incubator. Peptides were solubilized in HBSS, HEPES &0.1% BSA buffer, and serially-diluted 10-fold, from 50 uM to 5 pM, intriplicate. FLIPR Tetra was used to add peptide to the cells with dye(1:5, for final peptide concentrations ranging from 10 uM to 1 pM).FLIPR dye inside the cells emitted fluorescence after binding tocalcium, while fluorescence from outside the cells was masked.Fluorescence was measured using 470-495 excitation and 515-575 emissionwavelengths on the FLIPR Tetra. Readings were done for 3 minutes total,beginning 10 seconds before the peptide addition. Maximum-minimum valueswere calculated and plotted for each peptide concentration, and GraphPadprism software was used to calculate EC₅₀ values at the curve inflectionpoints, for calcium flux stimulation by peptides.

Plasma Stability Assay: Materials:

Working solution: 1 mg/mL test article is prepared in Milli-Q waterExtraction solution: Methanol:Acetonitrile:Water (1:1:1) with 0.1%Formic Acid and 400 ng/mL Glyburide.Plasma: Male Sprague-Dawley rat plasma (with sodium heparin), purchasedfrom Bioreclamation LLC (Liverpool, N.Y.).Whole blood: Male Sprague Dawley whole blood (with sodium heparin),purchased from Bioreclamation LLC (Liverpool, N.Y.)Lung homogenate: Male rat Sprague Dawley lung was purchased fromBioreclamation LLC (Liverpool, N.Y.). The lung was homogenized usingpolytron homogenizer after addition of 5× volume of 1×PBS. Thehomogenate was centrifuged at 9000 rpm for 10 min at 4° C. Thesupernatant was centrifuged again at 3000 rpm for 30 min to make a clearsupernatant. Protein concentration was determined using a commercial kit(Pierce, Thermo Scientific).

Sample Preparation Procedure: (Peptides)

Test article was prepared in one of the following biological matrices:heparinized rat plasma, heparinized rat whole blood or lung homogenate.The plasma and whole blood sample was prepared at 5000 ng/mL by adding 5uL of 1 mg/mL Working solution to 995 uL of rat plasma or whole blood.Lung homogenate samples were prepared by diluting lung homogenate to 1mg/ml protein concentration with phosphate buffered saline (PBS),followed by addition of 5 uL Working solution to 995 uL diluted lunghomogenate. The samples were incubated at 37° C. with gentle shaking(65-75 rpm) in a water bath incubator. At times 0 min, 5 min, 15 min, 30min, 60 min, 120 and 240 min, 25 uL aliquots of incubation samples weretransferred to 96-well plate and immediately protein precipitated using150 uL of Extraction solution. After completion of incubationexperiment, the sample plate was centrifuged at 4000 rpm at 4° C. for 10minutes. Afterwards, a pipetting device (Tecan Temo) was used totransfer the supernatants to another plate and add 50 uL of water to allsamples. The plate was vortexed prior to LC-MS analysis.

Sample Preparation Procedure (Conjugates)

Test article is prepared at 50,000 ng/mL by adding 5 uL of 1 mg/mLWorking solution to 495 uL of rat plasma. The samples are incubated at37° C. with gentle shaking (65-75 rpm) in a water bath incubator. Attimes 0 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 and 24 hr, 50 uL aliquots ofincubation samples are transferred to 96-well plate and 100 uL 40 mMTCEP (tris(2-carboxyethyl)phosphine) are added to each sample. Thereaction mixture is incubated at 37° C. for 1 hour. After completion ofreaction, protein precipitation is performed using 300 uL ofacetonitrile. The sample plate is centrifuged at 4000 rpm at 4° C. for10 minutes. Afterwards, a pipetting device (Tecan Temo) is used totransfer 125 uL supernatants to another plate and adds 50 uL of water toall samples. The plate is vortexed prior to LC-MS analysis.

LC-MS Analysis of Stability Samples

HPLC: Agilent 1290 HPLC with autosamplerColumn: MAC-MOD ACE C18, 3 μm, 30 mm×2.1 mm i.d.Mobile phase A: 0.1% Formic acid in acetonitrileMobile phase B: 0.1% Formic acid in water

Gradient Program:

Time (min) Flow (mL) Mobile Phase A (%) Mobile Phase B (%) 0 0.4 95 50.5 0.4 95 5 1.5 0.4 5 95 4.1 0.4 5 95 4.2 0.4 95 5 5 0.4 95 5Mass spectrometer: Agilent Q-TOF 6530Data acquisition mode: Full scan with mass range of 100-1000 m/zData acquisition and analysis software: MassHunter

Data Analysis:

Stability assay: stability half-life, (t½), values were determined byconverting peak areas at each time point to percent remaining relativeto initial (t=0) peak area.

Percent remaining=100×(sample peak area)÷(t=0 peak area)

The natural log of percent remaining values were calculated and plottedagainst sample time (Microsoft Excel). The slope of this line, k, wasdetermined by linear regression (Microsoft Excel).Stability half-life was then calculated by the formula, t½=0.693÷k

Surrogate Activity-Based Plasma Stability Assay:

The calcium flux protocol described above was followed, with thefollowing changes. The peptides were also incubated with 5% rat plasma(Bioreclamation #RATPLNAHP-M, Na Heparin-treated). Readings were takenat time points t₀ and t₂₄ hrs, after incubation in a 37° C. tissueculture incubator. Peptide plasma half-life in minutes was estimated bycalculating the following:

1) LN((EC₅₀ at t₀)/(EC₅₀ at t_(24 hrs))),

2) Calculate slope of value above and

3) t_(1/2)=0.693/(slopê2).

Using the test assay (as described above) polypeptides of the inventionexhibited efficacy and stability in accordance to Tables 2 and 3,provided infra.

TABLE 2 Activity and stability of polypeptides Surrogate hAPJ Ca²⁺activity-based Flux EC₅₀ Plasma stability Peptide [nM] t½ [min] Example1 9.4 8 Example 2 3.2 39 Example 3 64.5 n.d. Example 4 74.6 506 Example5 1.6 5.0 Example 6 21.4 5 Example 7 11.8 9 Example 8 13.1 662.5 Example9 23.4 109.2 Example 10 46.9 57.4 Example 11 32.2 32.8 Example 12 21.410 Example 13 12.2 10 Example 14 48.7 7.5 Example 15 193.5 17.7 Example16 14.1 7 Example 17 410.3 11.4 Example 18 174 50 Example 19 79.3 50Example 20 1.0 11.9 Example 21 2.9 >1000 Example 22 3.3 >1000 Example 2372.2 3157 Example 24 85.8 1699 Example 25 227 >1000 Example 26 2292 52Example 27 86.5 82 Example 28 3.1 13 Example 29 4.2 705.0 Example 30 0.4660.7 Example 31 0.5 43.1 Example 32 3.0 >1000 Example 33 1.9 >1000Example 34 0.6 >1000 Example 35 2.0 155.0 Example 35 66.7 8 Example 364.2 34 Example 37 937 47 Example 38 175 41 Example 40 2479 >1000 Example41 839 >1000 Example 42 16.3 >1000 Example 43 28.9 >1000 Comparative 1.85.0 Example: Pyr1-apelin-13

TABLE 3 Correlation between plasma stability assay and surrogateactivity based plasma stability assay: Plasma Surrogate Activitystability based Plasma Peptide t½ [min] stability t½ [min] Pyr-1-Apelin13 6.6 5.0 Ex 32 377 >1000

The polypeptide of the present invention or bioconjugates thereof mayhave an APJ receptor potency similar to apelin-13 or pyr-1-apelin-13. Inone embodiment the polypeptide of the present invention or bioconjugatethereof has an EC₅₀ of less than 100 nM. In another embodiment thepolypeptide of the invention, or bioconjugate thereof, has an EC₅₀ ofless than 50 nM, preferably less than 25 nM and more preferably lessthan 15 nM. In yet another embodiment, the polypeptide of the presentinvention or a bioconjugate thereof has an EC₅₀ of less than 10 nM.

The polypeptide of the present invention, or bioconjugate thereof, mayhave plasma stability superior to apelin-13 or pyr-1-apelin-13. In oneembodiment, the plasma stability improvement is at least 2 fold. In oneembodiment, the polypeptide of the invention, or a bioconjugate thereof,has a plasma stability of at least 30 minutes. In another embodiment,the polypeptide of the invention, or a bioconjugate thereof, has aplasma stability of at least 60 minutes, or at least 80 min, preferablyat least 100 minutes and more preferably at least 150 minutes.

The polypeptide of the present invention, or a bioconjugate thereof, maybe administered either simultaneously with, or before or after, one ormore other therapeutic agent. The polypeptide or bioconjugate of thepresent invention may be administered separately, by the same ordifferent route of administration, or together in the samepharmaceutical composition as the other agents.

In one embodiment, the invention provides a product comprising apolypeptide of anyone of formulae I to IV, or an amide, an ester of asalt thereof, or a bioconjugate thereof, and at least one othertherapeutic agent as a combined preparation for simultaneous, separateor sequential use in therapy. In one embodiment, the therapy is thetreatment of a disease or condition responsive to the activation of theAPJ receptor.

Products provided as a combined preparation include a compositioncomprising a polypeptide of anyone of formulae I to IV, or an amide, anester of a salt thereof, or a bioconjugate thereof, and the othertherapeutic agent(s) together in the same pharmaceutical composition, ora polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof, or a bioconjugate thereof, and the other therapeuticagent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical compositioncomprising a polypeptide of anyone of formulae I to IV, or an amide, anester or a salt thereof, or a bioconjugate thereof and anothertherapeutic agent(s). Optionally, the pharmaceutical composition maycomprise a pharmaceutically acceptable excipient, as described above.

In one embodiment, the invention provides a kit comprising two or moreseparate pharmaceutical compositions, at least one of which contains apolypeptide of anyone of formula I to IV, or an amide, an ester or asalt thereof or a bioconjugate thereof. In one embodiment, the kitcomprises means for separately retaining said compositions, such as acontainer, divided bottle, or divided foil packet. An example of such akit is a blister pack, as typically used for the packaging of tablets,capsules and the like.

The kit of the invention may be used for administering different dosageforms, for example, oral and parenteral, for administering the separatecompositions at different dosage intervals, or for titrating theseparate compositions against one another. To assist compliance, the kitof the invention typically comprises directions for administration.

In the combination therapies of the invention, the peptide orbioconjugate of the invention and the other therapeutic agent may bemanufactured and/or formulated by the same or different manufacturers.Moreover, the peptide or bioconjugate of the invention and the othertherapeutic may be brought together into a combination therapy: (i)prior to release of the combination product to physicians (e.g. in thecase of a kit comprising the compound of the invention and the othertherapeutic agent); (ii) by the physician themselves (or under theguidance of the physician) shortly before administration; (iii) in thepatient themselves, e.g. during sequential administration of apolypeptide or bioconjugate of the invention and the other therapeuticagent.

Accordingly, the invention provides the use of a polypeptide of anyoneof formulae I to IV, or an amide, an ester or a salt thereof, or abioconjugate thereof, for treating a disease or condition responsive tothe agonism of the APJ receptor, wherein the medicament is prepared foradministration with another therapeutic agent. The invention alsoprovides the use of another therapeutic agent for treating a disease orcondition responsive to the agonism of the apelin receptor, wherein themedicament is administered with a polypeptide of anyone of formulae I toIV, or an amide, an ester or a salt thereof or a bioconjugate thereof.

The invention also provides a polypeptide of anyone of formulae I to IV,or a pharmaceutically acceptable salt thereof, or a bioconjugatethereof, for use in a method of treating a disease or conditionresponsive to the agonism of the APJ receptor, wherein the polypeptideof anyone of formulae I to IV, or an amide, an ester or a salt thereof,or bioconjugate thereof, is prepared for administration with anothertherapeutic agent. The invention also provides another therapeutic agentfor use in a method of treating a disease or condition responsive to theagonism of the APJ receptor, wherein the other therapeutic agent isprepared for administration with a polypeptide of anyone of formulae Ito IV, or an amide, an ester or a salt thereof, or a bioconjugatethereof. The invention also provides a polypeptide of anyone of formulaeI to IV, or an amide, an ester or a salt thereof, or a bioconjugatethereof, for use in a method of treating a disease or conditionresponsive to the agonism of the APJ receptor, wherein the polypeptideof anyone of formulae I to IV, or an amide, an ester or a salt thereof,or a bioconjugate thereof, is administered with another therapeuticagent. The invention also provides another therapeutic agent for use ina method of treating a disease or condition responsive to the agonism ofthe APJ receptor, wherein the other therapeutic agent is administeredwith a polypeptide of anyone of formulae I to IV or an amide, an esteror a salt thereof or a bioconjugate thereof.

The invention also provides the use of a polypeptide of anyone offormulae I to IV, or an amide, an ester or a salt thereof, or abioconjugate thereof, for treating a disease or condition responsive tothe agonism of the APJ receptor, wherein the patient has previously(e.g. within 24 hours) been treated with another therapeutic agent. Theinvention also provides the use of another therapeutic agent fortreating a disease or condition responsive to the agonism of the APJreceptor, wherein the patient has previously (e.g. within 24 hours) beentreated with a polypeptide of anyone of formulae I to IV, or an amide,an ester or a salt thereof, or a bioconjugate thereof.

In one embodiment, the other therapeutic agent is selected frominotropes, beta adrenergic receptor blockers, HMG-Co-A reductaseinhibitors, angiotensin II receptor antagonists, angiotensin convertingenzyme (ACE) Inhibitors, calcium channel blockers (CCB), endothelinantagonists, renin inhibitors, diuretics, ApoA-I mimics, anti-diabeticagents, obesity-reducing agents, aldosterone receptor blockers,endothelin receptor blockers, aldosterone synthase inhibitors (ASI), aCETP inhibitor, anti-coagulants, relaxin, BNP (nesiritide) and a NEPinhibitor.

The term “in combination with” a second agent or treatment includesco-administration of the polypeptide or bioconjugate of the invention(e.g., a polypeptide according to anyone of Formulae I-IV or apolypeptide otherwise described herein) with the second agent ortreatment, administration of the compound of the invention first,followed by the second agent or treatment and administration of thesecond agent or treatment first, followed by the peptide to bioconjugateof the invention.

The term “second agent” includes any agent which is known in the art totreat, prevent, or reduce the symptoms of a disease or disorderdescribed herein, e.g. a disorder or disease responsive to theactivation of the APJ receptor, such as for example, acute decompensatedheart failure (ADHF), chronic heart failure, pulmonary hypertension,atrial fibrillation, Brugada syndrome, ventricular tachycardia,atherosclerosis, hypertension, restenosis, ischemic cardiovasculardiseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention,diabetes (including gestational diabetes), obesity, peripheral arterialdisease, cerebrovascular accidents, transient ischemic attacks,traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries(including sunburn) and preeclampsia.

Examples of second agents include inotropes, beta adrenergic receptorblockers, HMG-Co-A reductase inhibitors, angiotensin II receptorantagonists, angiotensin converting enzyme (ACE) Inhibitors, calciumchannel blockers (CCB), endothelin antagonists, renin inhibitors,diuretics, ApoA-I mimics, anti-diabetic agents, obesity-reducing agents,aldosterone receptor blockers, endothelin receptor blockers, aldosteronesynthase inhibitors (ASI), a CETP inhibitor, anti-coagulants, relaxin,BNP (nesiritide) and/or a NEP inhibitor.

Inotropes as used herein include for example dobutamine, isoproterenol,milrinone, amirinone, levosimendan, epinephrine, norepinephrine,isoproterenol and digoxin.

Beta adrenergic receptor blockers as used herein include for exampleacebutolol, atenolol, betaxolol, bisoprolol, carteolol, metoprolol,nadolol, propranolol, sotalol and timolol.

Anti-coagulants as used herein include Dalteparin, Danaparoid,Enoxaparin, Heparin, Tinzaparin, Warfarin.

The term “HMG-Co-A reductase inhibitor” (also calledbeta-hydroxy-beta-methylglutaryl-co-enzyme-A reductase inhibitors)includes active agents that may be used to lower the lipid levelsincluding cholesterol in blood. Examples include atorvastatin,cerivastatin, compactin, dalvastatin, dihydrocompactin, fluindostatin,fluvastatin, lovastatin, pitavastatin, mevastatin, pravastatin,rosuvastatin, rivastatin, simvastatin, and velostatin, or,pharmaceutically acceptable salts thereof.

The term “ACE-inhibitor” (also called angiotensin converting enzymeinhibitors) includes molecules that interrupt the enzymatic degradationof angiotensin I to angiotensin II. Such compounds may be used for theregulation of blood pressure and for the treatment of congestive heartfailure. Examples include alacepril, benazepril, benazeprilat,captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat,fosinopril, imidapril, lisinopril, moexipril, moveltopril, perindopril,quinapril, ramipril, spirapril, temocapril, and trandolapril, or,pharmaceutically acceptables salt thereof.

The term “endothelin antagonist” includes bosentan (cf. EP 526708 A),tezosentan (cf. WO 96/19459), or, pharmaceutically acceptable saltsthereof.

The term “renin inhibitor” includes ditekiren (chemical name:[1S-[1R*,2R*,4R*(1R*,2R*)]]-1-[(1,1-dimethylethoxy)carbonyl]-L-prolyI-L-phenylalanyl-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2-methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-N-alfa-methyl-L-histidinamide);terlakiren (chemical name:[R—(R*,S*)]—N-(4-morpholinylcarbonyl)-L-phenylalany-N-[1-(cyclohexylmethyl)-2-hydroxy-3-(1-methylethoxy)-3-oxopropyl]-S-methyl-L-cysteineamide);Aliskiren (chemical name:(2S,4S,5S,7S)-5-amino-N-(2-carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3-methoxypropoxy) phenyl]methyl}-8-methyl-2-(propan-2-yl)nonanamide) andzankiren (chemical name:[1S-[1R*[R*(R*)],2S*,3R*]]—N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5-methylhexyl]-alfa-[[2-[[(4-methyl-1-piperazinyl)sulfonyl]methyl]-1-oxo-3-phenylpropyl]-amino]-4-thiazolepropanamide),or, hydrochloride salts thereof, or, SPP630, SPP635 and SPP800 asdeveloped by Speedel, or RO 66-1132 and RO 66-1168 of Formula (A) and(B):

pharmaceutically acceptable salts thereof.

The term “aliskiren”, if not defined specifically, is to be understoodboth as the free base and as a salt thereof, especially apharmaceutically acceptable salt thereof, most preferably ahemi-fumarate salt thereof.

The term “calcium channel blocker (CCB)” includes dihydropyridines(DHPs) and non-DHPs (e.g., diltiazem-type and verapamil-type CCBs).Examples include amlodipine, Bepridil, Diltiazem, felodipine, ryosidine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, nitrendipine, Verapamil andnivaldipine, and is preferably a non-DHP representative selected fromthe group consisting of flunarizine, prenylamine, diltiazem, fendiline,gallopamil, mibefradil, anipamil, tiapamil and verapamil, or,pharmaceutically acceptable salts thereof. CCBs may be used asanti-hypertensive, anti-angina pectoris, or anti-arrhythmic drugs.

The term “diuretic” includes thiazide derivatives (e.g., chlorothiazide,hydrochlorothiazide, methylclothiazide, and chlorothalidon).

The term “ApoA-I mimic” includes D4F peptides (e.g., formulaD-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F) (SEQ ID NO: 18)

An angiotensin II receptor antagonist or a pharmaceutically acceptablesalt thereof is understood to be an active ingredient which bind to theAT₁-receptor subtype of angiotensin II receptor but do not result inactivation of the receptor. As a consequence of the inhibition of theAT₁ receptor, these antagonists can, for example, be employed asantihypertensives or for treating congestive heart failure.

The class of AT₁ receptor antagonists comprises compounds havingdiffering structural features, essentially preferred are thenon-peptidic ones. For example, mention may be made of the compoundswhich are selected from the group consisting of valsartan, losartan,candesartan, eprosartan, irbesartan, saprisartan, tasosartan,telmisartan, the compound with the designation E-1477 of the followingformula

the compound with the designation SC-52458 of the following formula

and the compound with the designation ZD-8731 of the following formula

or, in each case, a pharmaceutically acceptable salt thereof.

Preferred AT₁-receptor antagonist are candesartan, eprosartan,irbesartan, losartan, telmisartan, valsartan. Also preferred are thoseagents which have been marketed, most preferred is valsartan or apharmaceutically acceptable salt thereof.

The term “anti-diabetic agent” includes insulin secretion enhancers thatpromote the secretion of insulin from pancreatic □-cells. Examplesinclude biguanide derivatives (e.g., metformin), sulfonylureas (SU)(e.g., tolbutamide, chlorpropamide, tolazamide, acetohexamide,4-chloro-N-[(1-pyrolidinylamino)carbonyl]-benzensulfonamide(glycopyramide), glibenclamide (glyburide), gliclazide,1-butyl-3-metanilylurea, carbutamide, glibonuride, glipizide,gliquidone, glisoxepid, glybuthiazole, glibuzole, glyhexamide,glymidine, glypinamide, phenbutamide, and tolylcyclamide), orpharmaceutically acceptable salts thereof. Further examples includephenylalanine derivatives (e.g., nateglinide[N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine] (cf. EP 196222and EP 526171) of the formula

repaglinide[(S)-2-ethoxy-4-{2-[[3-methyl-1-[2-(1-piperidinyl)phenyl]butyl]amino]-2-oxoethyl}benzoicacid] (cf. EP 589874, EP 147850 A2, in particular Example 11 on page 61,and EP 207331 A1); calcium(2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinlycarbonyl)-propionatedihydrate (e.g., mitiglinide (cf. EP 507534)); and glimepiride (cf. EP31058).

Further examples of second agents with which the peptide and polypeptideof the invention can be used in combination include DPP-IV inhibitors,GLP-1 and GLP-1 agonists.

DPP-IV is responsible for inactivating GLP-1. More particularly, DPP-IVgenerates a GLP-1 receptor antagonist and thereby shortens thephysiological response to GLP-1. GLP-1 is a major stimulator ofpancreatic insulin secretion and has direct beneficial effects onglucose disposal.

The DPP-IV (dipeptidyl peptidase IV) inhibitor can be peptidic or,preferably, non-peptidic. DPP-IV inhibitors are in each case genericallyand specifically disclosed e.g. in WO 98/19998, DE 196 16 486 A1, WO00/34241 and WO 95/15309, in each case in particular in the compoundclaims and the final products of the working examples, thesubject-matter of the final products, the pharmaceutical preparationsand the claims are hereby incorporated into the present application byreference to these publications. Preferred are those compounds that arespecifically disclosed in Example 3 of WO 98/19998 and Example 1 of WO00/34241, respectively.

GLP-1 (glucagon like peptide-1) is an insulinotropic protein which isdescribed, e.g., by W. E. Schmidt et al. in Diabetologia, 28, 1985,704-707 and in U.S. Pat. No. 5,705,483.

The term “GLP-1 agonists” includes variants and analogs ofGLP-1(7-36)NH₂ which are disclosed in particular in U.S. Pat. No.5,120,712, U.S. Pat. No. 5,118,666, U.S. Pat. No. 5,512,549, WO 91/11457and by C. Orskov et al in J. Biol. Chem. 264 (1989) 12826. Furtherexamples include GLP-1(7-37), in which compound the carboxy-terminalamide functionality of Arg³⁶ is displaced with Gly at the 37^(th)position of the GLP-1(7-36)NH₂ molecule and variants and analogs thereofincluding GLN⁹-GLP-1(7-37), D-GLN⁹-GLP-1(7-37), acetyl LYS⁹-GLP-1(7-37),LYS¹⁸-GLP-1(7-37) and, in particular, GLP-1(7-37)OH, VAL⁸-GLP-1(7-37),GLY⁸-GLP-1(7-37), THR⁸-GLP-1(7-37), MET⁸-GLP-1(7-37) and4-imidazopropionyl-GLP-1. Special preference is also given to the GLPagonist analog exendin-4, described by Greig et al. in Diabetologia1999, 42, 45-50.

Also included in the definition “anti-diabetic agent” are insulinsensitivity enhancers which restore impaired insulin receptor functionto reduce insulin resistance and consequently enhance the insulinsensitivity. Examples include hypoglycemic thiazolidinedione derivatives(e.g., glitazone,(S)-((3,4-dihydro-2-(phenyl-methyl)-2H-1-benzopyran-6-yl)methyl-thiazolidine-2,4-dione(englitazone),5-{[4-(3-(5-methyl-2-phenyl-4-oxazolyl)-1-oxopropyl)-phenyl]-methyl}-thiazolidine-2,4-dione(darglitazone),5-{[4-(1-methyl-cyclohexyl)methoxy)-phenyl]methyl}-thiazolidine-2,4-dione(ciglitazone),5-{[4-(2-(1-indolyl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(DRF2189),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy)]benzyl}-thiazolidine-2,4-dione(BM-13.1246), 5-(2-naphthylsulfonyl)-thiazolidine-2,4-dione (AY-31637),bis{4-[(2,4-dioxo-5-thiazolidinyl)methyl]phenyl}methane (YM268),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl}-thiazolidine-2,4-dione(AD-5075),5-[4-(1-phenyl-1-cyclopropanecarbonylamino)-benzyl]-thiazolidine-2,4-dione(DN-108)5-{[4-(2-(2,3-dihydroindol-1-yl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione,5-[3-(4-chloro-phenyl])-2-propynyl]-5-phenylsulfonyl)thiazolidine-2,4-dione,5-[3-(4-chlorophenyl])-2-propynyl]-5-(4-fluorophenyl-sulfonyl)thiazolidine-2,4-dione,5-{[4-(2-(methyl-2-pyridinyl-amino)-ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(rosiglitazone),5-{[4-(2-(5-ethyl-2-pyridyl)ethoxy)phenyl]-methyl}thiazolidine-2,4-dione(pioglitazone),5-{[4-((3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy)-phenyl]-methyl}-thiazolidine-2,4-dione(troglitazone),5-[6-(2-fluoro-benzyloxy)naphthalen-2-ylmethyl]-thiazolidine-2,4-dione(MCC555),5-{[2-(2-naphthyl)-benzoxazol-5-yl]-methyl}thiazolidine-2,4-dione(T-174) and5-(2,4-dioxothiazolidin-5-ylmethyl)-2-methoxy-N-(4-trifluoromethyl-benzyl)benzamide(KRP297)).

Further anti-diabetic agents include, insulin signalling pathwaymodulators, like inhibitors of protein tyrosine phosphatases (PTPases),antidiabetic non-small molecule mimetic compounds and inhibitors ofglutamine-fructose-6-phosphate amidotransferase (GFAT); compoundsinfluencing a dysregulated hepatic glucose production, like inhibitorsof glucose-6-phosphatase (G6Pase), inhibitors offructose-1,6-bisphosphatase (F-1,6-Bpase), inhibitors of glycogenphosphorylase (GP), glucagon receptor antagonists and inhibitors ofphosphoenolpyruvate carboxykinase (PEPCK); pyruvate dehydrogenase kinase(PDHK) inhibitors; inhibitors of gastric emptying; insulin; inhibitorsof GSK-3; retinoid X receptor (RXR) agonists; agonists of Beta-3 AR;agonists of uncoupling proteins (UCPs); non-glitazone type PPARγagonists; dual PPARα/PPARγ agonists; antidiabetic vanadium containingcompounds; incretin hormones, like glucagon-like peptide-1 (GLP-1) andGLP-1 agonists; beta-cell imidazoline receptor antagonists; miglitol;α₂-adrenergic antagonists; and pharmaceutically acceptable saltsthereof.

In one embodiment, the invention provides a combination, in particular apharmaceutical combination, comprising a therapeutically effectiveamount of the polypeptide according to the definition of anyone offormulae I to IV, or an amide, an ester, a salt thereof, or abioconjugate thereof, and one or more therapeutically active agentsselected from β-adrenergic receptor blockers such as acebutolol,atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol,sotalol and timolol; angiotensin II receptor antagonists such as AT1blockers; antidiabetic agents such as DPPIV inhibitors (e.g.vildagliptin) and GLP1 peptide agonist.

The term “obesity-reducing agent” includes lipase inhibitors (e.g.,orlistat) and appetite suppressants (e.g., sibutramine and phentermine).

An aldosterone synthase inhibitor or a pharmaceutically acceptable saltthereof is understood to be an active ingredient that has the propertyto inhibit the production of aldosterone. Aldosterone synthase (CYP11B2)is a mitochondrial cytochrome P450 enzyme catalyzing the last step ofaldosterone production in the adrenal cortex, i.e., the conversion of11-deoxycorticosterone to aldosterone. The inhibition of the aldosteroneproduction with so-called aldosterone synthase inhibitors is known to bea successful variant to treatment of hypokalemia, hypertension,congestive heart failure, atrial fibrillation or renal failure. Suchaldosterone synthase inhibition activity is readily determined by thoseskilled in the art according to standard assays (e.g., US 2007/0049616).

The class of aldosterone synthase inhibitors comprises both steroidaland non-steroidal aldosterone synthase inhibitors, the later being mostpreferred.

Preference is given to commercially available aldosterone synthaseinhibitors or those aldosterone synthase inhibitors that have beenapproved by the health authorities.

The class of aldosterone synthase inhibitors comprises compounds havingdiffering structural features. An example of non-steroidal aldosteronesynthase inhibitor is the (+)-enantiomer of the hydrochloride offadrozole (U.S. Pat. Nos. 4,617,307 and 4,889,861) of formula

or, if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. inUS2007/0049616, in particular in the compound claims and the finalproducts of the working examples, the subject-matter of the finalproducts, the pharmaceutical preparations and the claims are herebyincorporated into the present application by reference to thispublication. Preferred aldosterone synthase inhibitors suitable for usein the present invention include, without limitation4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-methylbenzonitrile;5-(2-chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid (4-methoxybenzyl)methylamide;4′-fluoro-6-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid butyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;5-(2-Chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 4-fluorobenzyl ester;5-(4-Cyano-2-trifluoromethoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid methyl ester;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 2-isopropoxyethyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methylbenzonitrile;4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;3-Fluoro-4-(7-methylene-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)benzonitrile;cis-3-Fluoro-4-[7-(4-fluoro-benzyl)-5,6,7,8-tetrahydro-imidazo[1,5-a]pyridin-5-yl]benzonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrileor in each case, the (R) or (S) enantiomer thereof; or if appropriable,a pharmaceutically acceptable salt thereof.

The term aldosterone synthase inhibitors also include compounds andanalogs disclosed in WO2008/076860, WO2008/076336, WO2008/076862,WO2008/027284, WO2004/046145, WO2004/014914, WO2001/076574.

Furthermore Aldosterone synthase inhibitors also include compounds andanalogs disclosed in U.S. patent applications US2007/0225232,US2007/0208035, US2008/0318978, US2008/0076794, US2009/0012068,US20090048241 and in PCT applications WO2006/005726, WO2006/128853,WO2006128851, WO2006/128852, WO2007065942, WO2007/116099, WO2007/116908,WO2008/119744 and in European patent application EP 1886695. Preferredaldosterone synthase inhibitors suitable for use in the presentinvention include, without limitation8-(4-Fluorophenyl)-5,6-dihydro-8H-imidazo[5,1-c1[1,41oxazine;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-fluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2,6-difluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-methoxybenzonitrile;3-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)phthalonitrile;4-(8-(4-Cyanophenyl)-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)naphthalene-1-carbonitrile;8-[4-(1H-Tetrazol-5-yl)phenyl]-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazineas developed by Speedel or in each case, the (R) or (S) enantiomerthereof; or if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. in WO2009/156462 and WO 2010/130796, in particular in the compound claims andthe final products of the working examples, the subject-matter of thefinal products, the pharmaceutical preparations and the claims.Preferred Aldosterone Synthase inhibitors suitable for combination inthe present invention include,3-(6-Fluoro-3-methyl-2-pyridin-3-yl-1H-indol-1-ylmethyl)-benzonitrilehydrochloride,1-(4-Methanesulfonyl-benzyl)-3-methyl-2-pyridin-3-yl-1H-indole,2-(5-Benzyloxy-pyridin-3-yl)-6-chloro-1-methyl-1H-indole,5-(3-Cyano-1-methyl-1H-indol-2-yl)-nicotinic acid ethyl ester,N-[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,Pyrrolidine-1-sulfonic acid5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl ester,N-Methyl-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,6-Chloro-1-methyl-2-{5-[(2-pyrrolidin-1-yl-ethylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,6-Chloro-2-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-pyridin-3-yl]-1-methyl-1H-indole-3-carbonitrile,6-Chloro-1-methyl-2-{5-[(1-methyl-piperidin-4-ylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,Morpholine-4-carboxylic acid[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-amide,N-[5-(6-Chloro-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,C,C,C-Trifluoro-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-4-trifluoromethyl-benzenesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-1-phenyl-methanesulfonamide,N-(5-(3-chloro-4-cyanophenyl)pyridin-3-yl)butane-1-sulfonamide,N-(1-(5-(4-cyano-3-methoxyphenyl)pyridin-3-yl)ethyl)ethanesulfonamide,N-((5-(3-chloro-4-cyanophenyl)pyridin-3-yl)(cyclopropyl)methyl)ethanesulfonamide,N-(cyclopropyl(5-(1H-indol-5-yl)pyridin-3-yl)methyl)ethanesulfonamide,N-(cyclopropyl(5-naphtalen-1-yl-pyridin-3-yl)methyl)ethanesulfonamide,Ethanesulfonic acid[5-(6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-pyridin-3-ylmethyl]-amideand Ethanesulfonic acid{[5-(3-chloro-4-cyano-phenyl)-pyridin-3-yl]-cyclopropyl-methyl}-ethyl-amide.

The term “endothelin receptor blocker” includes bosentan andambrisentan.

The term “CETP inhibitor” refers to a compound that inhibits thecholesteryl ester transfer protein (CETP) mediated transport of variouscholesteryl esters and triglycerides from HDL to LDL and VLDL. Such CETPinhibition activity is readily determined by those skilled in the artaccording to standard assays (e.g., U.S. Pat. No. 6,140,343). Examplesinclude compounds disclosed in U.S. Pat. No. 6,140,343 and U.S. Pat. No.6,197,786 (e.g.,[2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylicacid ethyl ester (torcetrapib); compounds disclosed in U.S. Pat. No.6,723,752 (e.g.,(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol);compounds disclosed in U.S. patent application Ser. No. 10/807,838;polypeptide derivatives disclosed in U.S. Pat. No. 5,512,548;rosenonolactone derivatives and phosphate-containing analogs ofcholesteryl ester disclosed in J. Antibiot., 49(8): 815-816 (1996), andBioorg. Med. Chem. Lett.; 6:1951-1954 (1996), respectively. Furthermore,the CETP inhibitors also include those disclosed in WO2000/017165,WO2005/095409, WO2005/097806, WO 2007/128568, WO2008/009435, WO2009/059943 and WO2009/071509.

The term “NEP inhibitor” refers to a compound that inhibits neutralendopeptidase (NEP) EC 3.4.24.11. Examples include Candoxatril,Candoxatrilat, Dexecadotril, Ecadotril, Racecadotril, Sampatrilat,Fasidotril, Omapatrilat, Gemopatrilat, Daglutril, SCH-42495, SCH-32615,UK-447841, AVE-0848, PL-37 and(2R,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoicacid ethyl ester or a pharmaceutically acceptable salt thereof. NEPinhibitors also include Phosphono/biaryl substituted dipeptidederivatives, as disclosed in U.S. Pat. No. 5,155,100. NEP inhibitorsalso include N-mercaptoacyl phenylalanine derivative as disclosed in PCTapplication Number WO 2003/104200. NEP inhibitors also includedual-acting antihypertensive agents as disclosed in PCT applicationNumbers WO 2008/133896, WO 2009/035543 or WO 2009/134741. Other examplesinclude compounds disclosed in U.S. application Ser. Nos. 12/788,794;12/788,766 and 12/947,029. NEP inhibitors also include compoundsdisclosed in WO 2010/136474, WO 2010/136493, WO 2011/061271 and U.S.provisional applications Nos. 61/414,171 and 61/414,163.

In one embodiment, the invention provides a method of activating the APJreceptor in a subject, wherein the method comprises administering to thesubject a therapeutically effective amount of the polypeptide accordingto the definition of anyone of formulae I to IV, or an amide, an esteror a salt thereof or a bioconjugate thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation of the APJ receptor,in a subject, wherein the method comprises administering to the subjecta therapeutically effective amount of the polypeptide according to thedefinition of anyone of formulae I to IV, or an amide, an ester or asalt thereof or a bioconjugate thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation (agonism) of the APJreceptor, in a subject, wherein the disorder or the disease is selectedfrom acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

In one embodiment, the invention provides a polypeptide according to thedefinition of anyone of formulae I to IV, or a bioconjugate thereof, foruse as a medicament.

In one embodiment, the invention provides the use of a polypeptideaccording to the definition of anyone of formulae I to IV, or an amide,an ester or a salt thereof, or a bioconjugate thereof, in themanufacture of a medicament, for the treatment of a disorder or diseaseresponsive to the activation of the APJ receptor. In another embodiment,the invention provides the use of a polypeptide according to thedefinition of anyone of formulae I to IV, or an amide, an ester or asalt thereof, or a bioconjugate thereof, in the manufacture of amedicament, for the treatment of a disorder or disease responsive to theactivation of the APJ receptor, wherein said disorder or disease is inparticular selected from acute decompensated heart failure (ADHF),chronic heart failure, pulmonary hypertension, atrial fibrillation,Brugada syndrome, ventricular tachycardia, atherosclerosis,hypertension, restenosis, ischemic cardiovascular diseases,cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes(including gestational diabetes), obesity, peripheral arterial disease,cerebrovascular accidents, transient ischemic attacks, traumatic braininjuries, amyotrophic lateral sclerosis, burn injuries (includingsunburn) and preeclampsia.

Exemplification of the Invention: Peptide and Polypeptide Synthesis

Abbreviation Definition AA Amino acid Ac Acetyl Acm Acetamidomethyl ACNAcetonitrile AcOH Acetic acid Ac₂O Acetic anhydride ε-Ahx ε-Aminohexanoic acid AM Aminomethyl BAL Backbone amide linker BSA Bovine SerumAlbumin Boc tert-Butyloxycarbonyl Bzl Benzyl DCM Dichlormethane DICN,N′-Diisopropylcarbodiimide DIPEA N,N′-Diisopropylethylamine DMAN,N′-Dimethylacetamide DMF N,N′-Dimethylformamide DMSO DimethylsulfoxideDTT Dithiothreitol DVB Divinylbenzene EDT Ethanedithiol FA Formic acidFmoc 9-Fluorenylmethyloxycarbonyl HATU2-(1H-9-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate HBSS Hank's buffered salt solution HCTU2-(6-Chloro-1H-Benzotriazole-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid HFIP Hexafluoroisopropanol HOAt 1-Hydroxy-7-azabenzotriazole HSAHuman serum albumin HPLC High performance liquid chromatography HRMSHigh resolution mass spectrometry ivDde(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LN Logarithmusnaturali (natural logarithm) MeOH Methanol MS Mass spectrometry Nal2-Naphthylalanine Nle Norleucine NMP N-Methylpyrrolidine Oxyma PureEthyl 2-cyano-2-(hydroxyimino)acetate Pbf2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl pE Pyroglutamate PGProtecting group PBS Phosphate buffered saline Ph Phenyl PhPPhenylproline Pip Pipecolic acid Pol Polymeric support PS Polystyrene rtRoom temperature SEC Size exclusion chromatography SPPS Solid phasepeptide synthesis TBME tert-Butylmethylether TCEPTris(2-carboxyethyl)phosphine tBuOH tert-Butanol TFA Trifluoroaceticacid THF Tetrahydrofuran TIS, TIPS Triisopropylsilane t_(R) Retentiontime Trt Trityl UPLC Ultra performance liquid chromatography UVUltraviolet

The peptides were synthesized by standard solid phase Fmoc chemistry.The peptides were assembled on the Prelude™ peptide synthesizer (ProteinTechnologies, Inc., Tucson, USA) and Liberty microwave peptidesynthesizer (CEM Corporation, North Carolina, USA). Peptides with a freecarboxylic acid on the C-terminus or with an N,N-disubstitutedcarboxamide on the C-terminus were synthesized from 2-chlorotritylchloride-PS-resin (ABCR, Karlsruhe, Germany or AnaSpec, Inc.,California, USA). Peptides with an unsubstituted carboxamide on theC-terminus were synthesized from Fmoc protected Rink-Amide-AM-PS-resin(Merck, Darmstadt, Germany). Peptides with an N-monosubstitutedcarboxamide on the C-terminus were synthesized from BAL-AM-PS-resinloaded with amines (EMC Microcollections, Tübingen, Germany).

The peptides were purified by preparative reversed-phase HPLC. Thefollowing columns were used:

-   -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×100 mm, Part No.        186002572 (one column or two columns in series)    -   Waters Atlantis Prep OBD T3 Column, 5 μm, 30×150 mm, Part No.        186003703    -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×50 mm, Part No.        186002572

Mobile phases consisted of eluent A (0.1% TFA in H₂O) and eluent B(ACN). Gradients were designed based on the specific requirements of theseparation problem. Pure products were lyophilized from ACN/H₂O.

The products were analyzed by analytical HPLC using UV detection atλ=214 nm and UPLC-MS using electrospray ionization.

The peptides that are exemplified in Table 4 were synthesized using thegeneral procedures described below. Unsubstituted N- or C-termini areindicated by italic H— or —OH, respectively.

TABLE 4 Example Sequence SEQ ID NO: Example 1Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-F-OH 19 Example 2Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NH(Phenethyl) 20 Example 3Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-(N-Me)F-OH 21 Example 4Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NH ₂ 22 Example 5Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-Nal-OH 23 Example 6Ac-C*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OH 24 Example 7Ac-hC*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OH 25 Example 8Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-a-OH 26 Example 9Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NMe(Phenethyl) 27 Example 10Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-f-OH 28 Example 11Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-NH(Phenethyl) 29 Example 12Ac-c*-R-P-R-L-S-C*-K-G-P-Nle-P-F-OH 30 Example 13Ac-c*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OH 31 Example 14Ac-c*-R-P-R-L-S-c*-K-G-P-Nle-P-F-OH 32 Example 15Ac-C*-R-P-R-L-S-c*-K-G-P-Nle-P-F-OH 33 Example 16Ac-(D-hC)*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OH 34 Example 17Ac-(D-hC)*-R-P-R-L-S-(D-hC)*-K-G-P-Nle-P-F-OH 35 Example 18Ac-(D-hC)*-R-P-R-L-S-(hC)*-K-G-P-f-a-f-OH 36 Example 19Ac-c-R-P-R-L-S-(hC)-K-G-P-(D-Nle)-a-f-OH 37 Example 20pE-R-C*-R-L-S-C*-K-G-P-Nle-P-F-OH 38 Example 21pE-R-C*-R-L-S-C*-K-G-P-(D-Nle)-a-f-OH 39 Example 22pE-R-C*-R-L-S-C*-F-G-P-(D-Nle)-a-f-OH 40 Example 23pE-R-C*-R-L-S-C*-K-a-P-(D-Nle)-a-f-OH 41 Example 24pE-R-C*-R-L-S-C*-K-A-P-(D-Nle)-a-f-OH 42 Example 25pE-R-C*-R-L-S-C*-K-G-P-(D-Nle)-NH ₂ 43 Example 26pE-R-C*-R-L-S-(D-hC)*-K-G-P-f-a-f-OH 44 Example 27pE-R-c*-R-L-S-C*-K-G-P-(D-Nle)-(D-abu)-f-OH 45 Example 28pE-R-P-C*-L-S-C*-K-G-P-Nle-P-F-OH 46 Example 29pE-R-P-C*-L-S-C*-K-G-P-Nle-P-(N-Me)F-OH 47 Example 30pE-R-P-C*-L-S-C*-K-G-P-Nle-P-NH(Phenethyl) 48 Example 31pE-R-P-C*-L-S-C*-K-G-P-Nle-NH(Phenethyl) 49 Example 32pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) 50 Example 33pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-a-f-OH 51 Example 34pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-f-OH 52 Example 35pE-R-P-C*-L-S-C*-F-G-P-(D-Nle)-a-f-OH 53 Example 35H-R-P-C*-L-S-C*-K-(D-Nle)-a-f-OH 54 Example 36pE-R-P-hC*-L-S-C*-K-G-P-f-a-f-OH 55 Example 37pE-R-P-c*-L-S-(D-hC)*-K-G-P-(D-Nle)-a-y-OH 56 Example 38pE-R-P-(D-hC)*-L-S-hC*-K-G-P-(D-Nle)-(D-Nva)-f-OH 57wherein the two amino acids labeled with “*” represent the amino acidsforming a disulfide or amide bond via their side chain.

Analytical Methods 1a) HPLC—Analytical Method A

-   -   Column: Waters Xbridge™ C18 (50×4.0 mm), 3.5 μm; Part no:        186003031    -   Eluent A: 0.07% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.5 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 95 5 10.0 0 100 12.0 0 100 12.2 95 5

1b) HPLC—Analytical Method B

-   -   Column: XBridge BEH300 C18 (100×4.6 mm), 3 μm; Part no:        186003612    -   Eluent A: 0.1% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 98 2 18 2 98 20 2 98 22 98 2

2a) UPLC-MS—Analytical Method C

-   -   Column: Acquity UPLC® BEH300 C18 (50×2.1 mm), 1.7 μm; Part no:        186003685    -   Eluent A: 0.05% TFA in water/Eluent B: 0.04% TFA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 80° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 100 0 0.2 100 0 4.4 2 98 4.8 2 98 4.9 100 05.0 100 0

2b) UPLC-HRMS—Analytic Method D

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part no:        186002350    -   Eluent A: 0.05% FA+3.75 mM ammonium acetate in water; Eluent B:        0.04% FA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 50° C.    -   Gradient: 2 to 98% in 4.4 min

3) Analytical Method E:

-   -   XBridge C18 Column, 3.5 μm, 3.0×30 mm    -   Eluent: A: Water (0.1% formic acid); B: CAN    -   Flow rate: 2 mL/min    -   Gradient: 0 min 40% B; 40% to 95% B in 1.70 min; 2.0 min 95% B;        2.1 min 40% B    -   Mass Spectrometer: Single Quadrupole ESI scan range 150-1600    -   HPLC: Agilent 1100 series    -   Temperature: 40 C

3) Analytical Method F:

-   -   Acquity BEH 1.7 μm 2.1×50 mm    -   Eluent: A: Water (0.1% formic acid); B: ACN (0.1% formic acid)    -   Flow rate: 1 mL/min    -   Gradient: 0 min 2% B; 2% to 98% B in 1.7 min; 2.06 min 98% B;        2.16 min 2% B    -   Mass Spectrometer: Single Quadrupole ESI scan range 120-1600    -   HPLC: waters Acquity    -   Temperature: 50 C

3) Analytical Method G:

-   -   Acquity BEH 1.7 μm 2.1×50 mm    -   Eluent: A: Water (0.1% formic acid); B: ACN (0.1% formic acid)    -   Flow rate: 1 mL/min    -   Gradient: 0 min 40% B; 40% to 98% B in 1.40 min; 2.05 min 98% B;        2.1 min 40% B    -   Mass Spectrometer: Single Quadrupole ESI scan range 120-1600    -   HPLC: waters Acquity    -   Temperature: 50 C        The analytical data for peptides of Examples 1 to 38 are        summarized in Table 5 and was generated using the analytical        methods described supra.

TABLE 5 HPLC Mass spectrometry t_(R) [M + 2H]²⁺ [M + 3H]³⁺ [M + 2H]²⁺[M + 3H]³⁺ Peptide [min] Meth. (measured) (measured) Meth. (calc.)(calc.) Example 1 3.48 A 757.4 505.3 C 757.4 505.3 Example 2 3.66 A735.4 490.6 C 735.4 490.6 Example 3 3.60 A 764.4 509.9 C 764.4 509.9Example 4 2.65 A 683.4 455.9 C 683.4 455.9 Example 5 3.95 A 782.4 521.9C 782.4 521.9 Example 6 3.49 A 764.4 509.9 C 764.4 509.9 Example 7 3.55A 771.4 514.6 C 771.4 514.6 Example 8 2.85 A 719.3 479.9 C 719.4 479.9Example 9 3.75 A 742.4 495.3 C 742.4 495.3 Example 10 3.55 A 757.4 505.3C 757.4 505.3 Example 11 3.57 A 686.9 458.3 C 686.9 458.2 Example 123.51 A 757.4 505.3 C 757.4 505.3 Example 13 3.54 A 764.4 509.9 C 764.4509.9 Example 14 3.46 A 757.4 505.3 C 757.4 505.3 Example 15 3.44 A757.4 505.3 C 757.4 505.3 Example 16 3.59 A 771.4 514.6 C 771.4 514.6Example 17 3.61 A 771.4 514.6 C 771.4 514.6 Example 18 7.60 B 775.391517.263 D 775.385 517.256 Example 19 7.40 B 751.392 501.263 D 751.385501.256 Example 20 3.41 A 743.4 495.9 C 743.4 495.9 Example 21 7.35 B730.355 487.239 D 730.361 487.241 Example 22 8.62 B 739.877 493.587 D739.856 493.573 Example 23 7.32 B 737.374 491.918 D 737.377 491.921Example 24 7.35 B 737.375 491.919 D 737.377 491.921 Example 25 5.63 B620.822 414.217 D 620.825 414.219 Example 26 7.58 B 754.368 503.247 D754.361 503.241 Example 27 7.36 B 737.374 491.918 D 737.369 491.913Example 28 3.63 A 713.9 476.2 C 713.8 476.2 Example 29 3.78 A 720.9480.9 C 720.9 480.9 Example 30 3.82 A 691.9 461.6 C 691.8 461.6 Example31 3.74 A 643.3 429.2 C 643.3 429.2 Example 32 8.01 B 643.328 429.222 D643.324 429.252 Example 33 7.73 B 700.832 467.557 D 700.845 467.566Example 34 7.17 B 609.809 406.875 D 609.810 406.876 Example 35 8.99 B710.351 D 710.332 473.890 Example 35 6.89 B 568.289 379.196 D 568.292379.197 Example 36 7.69 B 724.843 483.564 D 724.837 483.558 Example 376.62 B 715.848 477.568 D 715.843 477.562 Example 38 8.28 B 728.873 D728.869

General Synthesis Procedures

1) Loading of First Amino Acid onto 2-Chlorotrityl Chloride Resin andFmoc-Removal

2-Chlorotrityl chloride resin (1 eq., 1.0-1.6 mmol/g) was washedthoroughly with DCM. The desired amino acid (typically 0.5-2 eq.relative to the resin, considering 1.6 mmol/g loading) was dissolved inDCM (approx. 10 mL per gram of resin) and DIPEA (4 eq. relative to theresin, considering 1.6 mmol/g loading). The solution was added to theresin and the suspension was shaken at rt for 3-16 h. The resin wasdrained and then thoroughly washed sequentially with DCM/MeOH/DIPEA(17:2:1), DCM, DMA, DCM.

For Fmoc removal and determination of the loading the resin was shakenrepeatedly with piperidine/DMA (1:4) or 4-methylpiperidine/DMA (1:4)(12×10 mL per gram of initial resin) and washed with DMA (2×10 mL pergram of initial resin). The combined solutions were diluted with MeOH toa volume V of 250 mL per gram of initial resin. A 2 mL aliquot (V_(a))of this solution was diluted further to 250 mL (V_(t)) with MeOH. The UVabsorption was measured at 299.8 nm against a reference of MeOH, givingabsorption A. The resin was thoroughly washed sequentially with DMA,DCM, DMA, DCM and dried in high vacuum at 40° C., affording m g ofresin.

The loading of the resin is calculated according to the formula:

Loading [mol/g]=(A×V _(t) ×V)/(d×ε×V _(a) ×m)

(with d: width of cuvette; ε=7800 L mol⁻¹ cm⁻¹)

2) Solid Phase Peptide Synthesis 2a) Synthesis Cycle A on Prelude™Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4) or 4-methylpiperidine/DMA (1:4). The resin waswashed with DMA. Coupling was done by addition of the Fmoc-amino acid (3eq.; 0.3 M solution in NMP), HCTU (3 eq.; 0.3 M solution in NMP), andDIPEA (4.5 eq.; 0.9 M solution in NMP) followed by mixing of thesuspension with nitrogen at rt for typically 15 min to 4 h depending onthe specific requirements. After washing with DMA the coupling step wastypically repeated 1 to 3 times depending on the specific requirements.After washing with DMA capping was performed by addition of a mixture ofAc₂O/pyridine/DMA (1:1:8) and subsequent mixing of the suspension at rt.The resin was washed with DMA.

2a) Synthesis Cycle B on Prelude™ Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith 4-methylpiperidine/DMA (1:4). The resin was washed with DMA.Coupling was done by addition of the Fmoc-amino acid (3 eq.; 0.2 Msolution in NMP), HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (3.3eq.; 0.66 M solution in NMP) followed by mixing of the suspension withnitrogen at rt for typically 15 min to 4 h depending on the specificrequirements. After washing with DMA the coupling step was typicallyrepeated 1 to 3 times depending on the specific requirements. Afterwashing with DMA capping was performed by addition of a mixture ofAc₂O/pyridine/DMA (1:1:8) and subsequent mixing of the suspension at rt.The resin was washed with DMA.

2b) Synthesis Cycle C on Prelude™ Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4) or 4-methylpiperidine/DMA (1:4). The resin waswashed with DMA. Capping was performed by addition of a mixture ofAc₂O/pyridine/DMA (1:1:8) and subsequent mixing of the suspension at rt.The resin was washed with DMA.

2c) Synthesis Cycle D on Liberty™ Synthesizer

The resin was washed with DMF and DCM. Fmoc was removed by treatmentwith 20% piperidine/DMF (typically 7 ml per 0.1 mmol twice). The resinwas washed with DMF and DCM. Coupling was done by addition of theFmoc-amino acid (5 eq.; 0.2 M solution in DMF), HCTU (5 eq.; 0.5 Msolution in DMF), and DIPEA (10 eq.; 2 M solution in NMP) followed bymixing of the suspension with nitrogen at 75 or 50° C. for typically 5to 50 min with microwave power 0 to 20 watts depending on the specificrequirements. After washing with DMF the coupling step might be repeatedonce depending on the specific requirements. The resin was washed withDMF.

3) Cleavage from Resin with or without Concomitant Removal of ProtectingGroups

3a) Cleavage Method A

The resin (0.1 mmol) was shaken at rt for 2 h with 95% aq. TFA/EDT/TIS(95:2.5:2.5) (3 mL). The cleavage solution was filtered off, and freshsolution was added (3 mL). The suspension was shaken at rt for 1 h thenthe cleavage solution was filtered off. Fresh solution was added (3 mL)and the suspension was shaken at rt for 1 h. The cleavage solution wasfiltered off. The combined cleavage solutions were poured slowly onto amixture of cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (10-20mL), the suspension was centrifuged and the supernatant was poured off.This step was performed 1-2-times. The solid was dried in high vacuum.

3b) Cleavage Method B

The resin (0.1 mmol) was shaken at rt for 3 h with 95% aq. TFA//TIS/DTT(95:2.5:2.5) (3 mL). The cleavage solution was filtered off. The resinwas rinsed once with 95% aq. TFA (1 mL). The combined cleavage andwashing solutions were poured slowly onto a mixture of coldheptane/diethyl ether (1:1) (10-15 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. Diethyl ether(10 mL) was added to the residue, the suspension was vortexed for 3 minand centrifuged, and the supernatant was poured off, The wash processwas repeated twice. The solid was dried in high vacuum.

3c) Cleavage Method C

HFIP/DCM (1:3) (3 mL) was added to the resin (0.1 mmol) and thesuspension was shaken at rt for 20 min. The cleavage solution wasfiltered off and collected. This procedure was repeated twice. Finallythe resin was washed once with HFIP/DCM (1:3) (1 mL). The combinedcleavage and washing solutions were concentrated to dryness in vacuo.The crude product was lyophilized from ACN/H₂O.

4) Disulfide Formation 4a) Cyclization Method A

The fully deprotected linear precursor peptide was dissolved in H₂O/DMSO(9:1) or (4:1) to give typically a concentration of 0.5-7 mM. Thereaction mixture was then stirred at rt for typically 16-96 h dependingon the requirements and then concentrated to dryness in high vacuum.

4b) Cyclization Method B

The fully deprotected linear precursor peptide (1 eq.) was dissolved inH₂O to give typically a concentration of about 1-25 mM. A solution of 50mM I₂ in AcOH (1-2 eq.) was added and the mixture was stirred at rtuntil complete conversion is achieved. 0.5 M Ascorbic acid in H₂O wasadded to quench the excess of I₂.

In the following the syntheses of representative examples are described.

Example 9 Synthesis of Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NMe(Phenethyl)(Disulfide C¹-C⁷) (SEQ ID NO: 27)

(SEQ ID NOS 58, 58, 27, and 27, respectively, in order of appearance)

Preparation of Intermediate 9a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-P—OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (2.00 g, 3.20 mmol) was washed with DCM(3×). A solution of Fmoc-P—OH (1.08 g, 3.20 mmol) in DCM (20 mL) andDIPEA (2.24 mL, 12.8 mmol) was added and the suspension was shaken for 3h at rt. The resin was washed thoroughly with DCM/MeOH/DIPEA (17:2:1)(3×), DCM (3×), DMA (3×), DCM (3×).

The resin was then treated twelve times for 2 min with a mixture ofpiperidine/DMA (1:4) (12 mL each time). The piperidine/DMA solutionswere collected for determination of the loading of the resin (seegeneral procedure).

The resin was washed thoroughly with DCM (3×), DMA (3×), DCM (3×) anddried in vacuo to give Intermediate 9a (2.25 g; loading=1.12 mmol/g).

Preparation of Intermediate 9b

(Assembly of Linear Peptide)

Intermediate 9a (89 mg, 0.10 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 Nle 2× 1 h B 2 P 2 × 30 min B 3 G 2 × 1 h B 4 K(Boc) 2 × 30 min B 5 C(Trt) 2× 30 min B 6 S(tBu) 2 × 30 min B 7 L 2 × 30 min B 8 R(Pbf) 4 × 1 h B 9 P2 × 1 h B 10 R(Pbf) 4 × 1 h B 11 C(Trt) 2 × 1 h B 12 Ac 1 × 10 min C

Preparation of Intermediate 9c

(HFIP Cleavage from the Resin)

HFIP/DCM (1:3) (3 mL) was added to Intermediate 9b (0.100 mmol) and thesuspension was shaken at rt for 20 min. The cleavage solution wasfiltered off and collected. This procedure was repeated twice. Finallythe resin was washed once with HFIP/DCM (1:3) (1 mL). The combinedcleavage and washing solutions were concentrated to dryness in vacuo.The crude product was lyophilized from ACN/H₂O to afford Intermediate 9c(37.2 mg, 14.8 μmol) as a white solid.

Preparation of Intermediate 9d

(Amide Formation and Protecting Group Removal)

Intermediate 9c (37.0 mg, =14.7 μmol) and TBTU (7.1 mg, 22.1 μmol) weredissolved in DCM (5 mL). DIPEA (5.1 μl, 29.4 μmol) was added and thesolution was stirred for 2 min at rt. N-Methyl-phenethylamine (3.2 μl,22.1 μmol) was added, the reaction was stirred for 1 h 40 min at rt thenpartitioned between EtOAc/n-butanol (9:1) (50 mL) and 5% aq. NaHCO₃ (5mL). The organic layer was washed with 5% aq. NaHCO₃ (5 mL), brine (5mL), dried over Na₂SO₄, filtered and concentrated to dryness in vacuo.

The residue was dissolved in 95% aq. TFA/TIS/EDT (95:2.5:2.5) (5 mL).The reaction mixture was stirred for 3 h at rt then poured on coldheptane/ether (1:1) (35 mL). The suspension was centrifuged and thesolvent was decanted. The residue was washed twice with colddiethylether/heptane (1:1) (10 mL) and then dried in vacuo to affordIntermediate 9d (27.0 mg, 14.7 μmol) as a beige solid. The product wassubjected to the next step without purification.

Preparation of Example 9

(Disulfide Formation and Purification)

Intermediate 9d (26.9 mg, 14.7 μmol) was dissolved in H₂O (3.7 mL)(solution was slightly cloudy). A solution of 50 mM I₂ in AcOH (0.353mL, 17.6 μmol) was added and the mixture was stirred at rt for 30 min.0.5 M Ascorbic acid in H₂O (0.044 mL, 22.1 μmol) was added to quench theexcess of iodine. The solution was concentrated to about 3.5 mL and thensubjected to preparative reversed-phase HPLC. Fractions were lyophilizedto afford Example 9 (6.8 mg, 3.7 μmol) as a white solid.

The pure product was analyzed by analytical HPLC (Analytical method A;t_(R)=3.75 min) and UPLC-MS (Analytical method C; measured:[M+3]³⁺=495.3; calculated: [M+3]³⁺=495.3).

Example 12 Synthesis of Ac-c*-R-P-R-L-S-C*-K-G-P-Nle-P-F-OH (DisulfideC¹-C⁷) (SEQ ID NO: 30)

(SEQ ID NOS 59, 30, and 30, respectively, in order of appearance)

Preparation of Intermediate 12a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-F-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (10.0 g, 16.0 mmol) was washed with DCM(3×). A solution of Fmoc-F-OH (12.4 g, 32.0 mmol) in DCM (100 mL) andDIPEA (11.2 mL, 64.0 mmol) was added and the suspension was shaken for 5h at rt. The resin was washed thoroughly with DCM/MeOH/DIPEA (17:2:1)(3×), DCM (3×), DMA (3×), DCM (3×).

The resin was then treated twelve times for 2 min with a mixture ofpiperidine/DMA (1:4) (50 mL each time) followed by washing with DMA(2×). The piperidine/DMA solutions and DMA washing solutions werecollected for determination of the loading of the resin (see generalprocedure).

The resin was washed thoroughly with DCM (3×), DMA (3×), DCM (3×) anddried in vacuo to give Intermediate 12a (12.8 g; loading=0.79 mmol/g).

Preparation of Intermediate 12b

(Assembly of Linear Peptide)

Intermediate 12a (127 mg, 0.10 mmol) was subjected to solid phasepeptide synthesis on the Prelude™ peptide synthesizer. Coupling wasperformed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 P 2 ×1 h A 2 Nle 2 × 1 h A 3 P 2 × 1 h A 4 G 2 × 2 h A 5 K(Boc) 2 × 1 h A 6C(Trt) 1 × 3 h A 7 S(tBu) 2 × 1 h A 8 L 2 × 1 h A 9 R(Pbf) 4 × 3 h A 10P 2 × 2 h A 11 R(Pbf) 4 × 3 h A 12 c(Trt) 1 × 3 h A 13 Ac 1 × 20 min C

Preparation of Intermediate 12c

(Cleavage from the Resin with Concomitant Protecting Group Removal andPurification)

Intermediate 12b (0.10 mmol) was carefully washed with DCM (4×). Amixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL) was added and thesuspension was shaken at rt for 2 h. The cleavage solution was filteredoff, and fresh cleavage solution (2 mL) was added. The suspension wasshaken at rt for 1 h then the cleavage solution was filtered off. Freshsolution (2 mL) was added and the suspension was shaken at rt for 1 h.The cleavage solution was filtered off. The combined cleavage solutionswere poured onto a mixture of cold heptane/diethyl ether (1:1) (30 mL),giving a precipitate. The mixture was centrifuged and the supernatantpoured off. The solid was washed again with cold heptane/diethyl ether(1:1) (10 mL), the mixture was centrifuged and the supernatant pouredoff. The solid was dried in vacuo. The crude product was purified bypreparative HPLC and lyophilized to afford Intermediate 12c (53 mg,0.029 mmol).

Preparation of Example 12

(Cyclization and Purification)

Intermediate 12c (53 mg, 0.029 mmol) was dissolved in H₂O/DMSO (9:1) (18mL). The reaction mixture was stirred at rt for 40 h then concentratedto dryness in vacuo. The crude product was purified by preparative HPLCand lyophilized to afford Example 12 as a white solid (27.4 mg; 0.014mmol).

The pure product was analyzed by analytical HPLC (Analytical method A;t_(R)=3.51 min) and UPLC-MS (Analytical method C; measured:[M+3]³⁺=505.3; calculated: [M+3]³⁺=505.3).

Example 18 Synthesis of Ac-(D-hC)*-R-P-R-L-S-(hC)*-K-G-P-f-a-f-OH(Disulfide C¹-C⁷). (SEQ ID NO: 36) Linear Peptide Synthesis on SolidSupport:

Fmoc-D-Phe-Wang resin (substitution: 0.66 mmol/g) was subjected tomanual solid phase peptide synthesis via standard Fmoc chemistry. 0.3mmol resin was swelled in DMF for 30 minutes; DMF was drained and theresin was treated with 20% piperidine in DMF for 30 min to remove Fmocgroup. The resin was washed by DMF 3 times and coupled with apre-activated Fmoc amino acid solution (Fmoc aminoacid/HBTU/HOBt/NMM=3:3:3:6 eq) for 2 hours. Ninhydrin test was performedafter each coupling to check the coupling efficiency.

The peptide chain was assembled on resin by repetitive removal of theFmoc protecting group and coupling of protected amino acid till N-termend.

After the coupling of the last amino acid, peptide resin was washed withDMF and ethyl ether, and dried under vacuum. The dried peptide resin wastreated with TFA cleavage cocktail(TFA/thiolanisole/phenol/EDT/H₂O=87.5:5:2.5:2.5:2.5, v/v) for cleavageand removal of the side chain protecting groups. Crude peptides wereprecipitated from cold ether, collected by filtration and dried underhigh vacuum. Crude peptides was purified on HPLC (Column: 2″-inch DeltaPak C18, Wavelength: 215 nm) to afford desired product.

Cyclisation:

Each of crude peptides was dissolved in water-Acetonitrile (A.C.S.reagent, Fisher) at a concentration of 1 mg/mL (around 80%:20%; Water:Acetonitrile, V:V), 0.1 M I₂ (A.C.S. reagent, Sigma Aldrich) in 50% AcOH(A.C.S. reagent, Fisher)/H₂O was added drop-wise into the solution withvigorous stirring until I₂ color persist. Upon completion of oxidation(monitored by analytical HPLC and Mass spectroscopy), 1M L-ascorbic acid(A.C.S. reagent, Sigma Aldrich) aqueous solution was drop-wise addedwith continuous stirring to reduce excess I₂ until the solution becomescolorless. After filtration, the above solution was loaded onto 2-inchC18 column (detection at 215 nm), and purified by using TFA buffer(Buffer A, 0.1% TFA (A.C.S. grade, NuGeneration Technology, LLC) inwater; Buffer B, 100% acetonitrile), collected fractions with purityof >95% were lyophilized to dry.

Example 21 Synthesis of pE-R-C*-R-L-S-C*-K-G-P-(D-Nle)-a-f-OH (DisulfideC³-C⁷) (SEQ ID NO: 39)

(SEQ ID NOS 60, 39, and 39, respectively, in order of appearance)

Preparation of Intermediate 21a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-f-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (5.0 g, 8.01 mmol) was reacted with asolution of Fmoc-f-OH (3.10 g, 8.01 mmol) in DCM (50 mL) and DIPEA (5.59mL, 32.0 mmol) in analogy to the general procedure described above togive Intermediate 21a (5.87 g, loading=0.897 mmol/g).

Preparation of Intermediate 21b

(Assembly of Linear Peptide)

Intermediate 21a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Liberty™ microwave peptide synthesizer. Coupling wasperformed as follows:

Number of Cou- couplings × Temperature Microwave Synthesis pling AAReaction time ° C. power cycle 1 a 1 × 7.5 min 50 20 D 2 D-Nle 1 × 7.5min 50 20 D 3 P 1 × 7.5 min 50 20 D 4 G 1 × 7.5 min 50 20 D 5 K(Boc) 1 ×7.5 min 50 20 D 6 C(Trt)  1 × 2 min 50 0 D  1 × 4 min 50 25 7 S 1 × 7.5min 50 25 D 8 L 1 × 7.5 min 50 25 D 9 R(Pbf)  2 × 42 min 50 0 D 2 × 7.5min 50 25 10 C(Trt)  1 × 2 min 50 0 D  1 × 4 min 50 25 11 R(Pbf)  2 × 42min 50 0 D 2 × 7.5 min 50 25 12 pE 1 × 7.5 min 50 25 D

Preparation of Intermediate 21c

(Cleavage from the Resin with Concomitant Protecting Group Removal thenPurification)

A mixture of 95% aq. TFA/EDT/DTT (95:2.5:2.5) (2 mL) was added toIntermediate 21b (0.1 mmol). The suspension was shaken at rt for 2 h,then the cleavage solution was filtered off. The resin was then againtreated with 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL), shaked at rt for 1h, and filtered. The combined cleavage and washing solutions were pouredonto a mixture of cold heptane/diethyl ether (1:1) (11 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. Diethyl ether (10 mL) was added to the residue, the suspension wasvortexed for 3 min and centrifuged, and the supernatant was poured off,The washing process was repeated twice. The solid was dried in highvacuum The crude was purified by preparative HPLC and lyophilized fromACN/H₂O to afford Intermediate 21c (55 mg, 0.030 mmol).

Preparation of Example 21

(Cyclization and Purification)

Intermediate 21c (18 mg, 9.97 μmol) was dissolved in H₂O (1.0 mL). Asolution of 50 mM I₂ in AcOH (0.24 mL, 12 μmol) was added in one portionto the stirred solution and the solution was stirred overnight at rtuntil LCMS showed completion of the reaction. 0.5 M Ascorbic acid in H₂O(24 μmol, 12 μmol) was added to quench the excess of I₂. The crude waspurified by preparative HPLC and lyophilized from ACN/H₂O to affordExample 21 as a white solid (12 mg, 6.32 μmol).

The pure product was analyzed by analytical HPLC (Analytical method B;t_(R)=7.35 min) and UPLC-HRMS (Analytical method D; measured:[M+2H]²⁺=730.355; calculated: [M+2H]²⁺=730.361).

Example 32 Synthesis of pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl)(Disulfide C⁴-C⁷) Acetate (SEQ ID NO: 50)

(SEQ ID NOS 61, 61, 61, 50, and 50, respectively, in order ofappearance)

Preparation of Intermediate 32a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-D-Nle-OH, FmocRemoval and Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (50.0 g, 85.0 mmol) was suspended in ofDCM (400 mL) the suspension was stirred for 10 min and then the solventwas drained, the resin was washed with DCM (3×200 mL). Then a solutionof Fmoc-D-Nle-OH (24.0 g, 68.0 mmol) and DIPEA (96.5 ml, 552.5 mmol) inDCM (120.0 mL) was added to the resin, the suspension was flushed withnitrogen and stirred at rt for 5 min. Another portion of DIPEA (22.7 ml,127.5 mmol) was added and the reaction mixture was stirred at rtovernight.

The reaction mixture was drained and the resin was washed with DCM(3×250 mL) for 2 min each time. The resin was quenched with of a mixtureDCM/MeOH/DIPEA (70:15:15) (2×250 mL) for 10 min each time.

The Fmoc group was cleaved by treating the resin with piperidine/DMF(1:3) (1×300 mL) for 5 min. the resin was drained then (1×300 mL) for 15min, followed by washing steps: DMF (6×250 mL, 2 min each time),isopropanol (2×250 mL, 2 min each time) and TBME (6×250 mL, 2 min eachtime). The resin was dried under vacuum at 35° C. for 24 hours to affordIntermediate 32a (57.8 g, loading=1.08 mmol/g).

Preparation of Intermediate 32b

(Assembly of Linear Peptide)

Intermediate 32a (18.5 g, 20.0 mmol) was subjected to solid phasepeptide synthesis on an automatic peptide synthesizer (CSBIO536™). Acoupling cycle was defined as follows:

-   -   Amino acid coupling: AA (3.0 eq.), DIC (3.0 eq.), HOBt (3.0        eq.), DMF (see table below)    -   Washing: DMF (4×150 mL, 2 min each time).    -   Fmoc deprotection: Piperidine/DMF (1:3) (150 mL for 5 min then        150 mL for 15 min).    -   Washing: DMF (6×150 mL, 2 min each time).

Cou- Number of couplings × Coupling pling AA Reaction time Method 1Fmoc-L-Pro-OH 1 × 120 min DIC/HOBt 2 Fmoc-Gly-OH 1 × 120 min DIC/HOBt 3Fmoc-L-Lys(Boc)-OH 1 × 120 min DIC/HOBt 4 Fmoc-L-Cys(Trt)-OH 1 × 120 minDIC/HOBt 5 Fmoc-L-Ser(tBu)-OH 1 × 120 min DIC/HOBt 6 Fmoc-L-Leu-OH 1 ×120 min DIC/HOBt 7 Fmoc-L-Cys(Trt)-OH 1 × 120 min DIC/HOBt 8Fmoc-L-Pro-OH 1 × 120 min DIC/HOBt 9 Fmoc-L-Arg(Pbf)-OH 1 × 120 minDIC/HOBt 10 Boc-L-Pyr—OH 1 × 120 min DIC/HOBt

After the assembly of the peptide, the resin was washed with DMF (6×150mL, 2 min each time), isopropanol (6×150 mL, 2 min each time) and TBME(6×150 mL, 2 min each time). The peptide resin was dried overnight underhigh vacuum at 35° C. to give Intermediate 32b (57.6 g, 20.0 mmol).

Preparation of Intermediate 32c

(HFIP Cleavage from the Resin)

A portion of Intermediate 32b (27 g, 9.37 mmol) was suspended in DCM(300 mL) and stirred for 15 min. The resin was drained then treated withHFIP/DCM (3:7) (3×270 mL, 15 min each time). The cleavage solution wasfiltered off and collected. The resin was washed with DCM (3×300 mL).The combined cleavage and washing solutions were concentrated to drynessin vacuo. The white powder was dried overnight under vacuum at 35° C.yielding Intermediate 32c-Batch1 (23.5 g, 9.37 mmol).

The above mentioned procedure was repeated with another portion ofIntermediate 32b (28.0 g, 9.72 mmol), affording Intermediate 32c-Batch2(26.1 g, 9.72 mmol).

Preparation of Intermediate 32d

(Solution Phase Coupling of Phenethylamine)

Intermediate 32c-Batch2 (20.0 g, 7.44 mmol, 1.0 eq) and HATU (5.23 g,13.8 mmol, 1.85 eq) were dissolved in DMF (400 mL). A solution ofphenethylamine (1.67 g, 13.8 mmol, 1.85 eq) and DIPEA (3.56 g, 27.6mmol, 3.71 eq) in DMF (60 mL) was added.

The reaction mixture was stirred at rt for 30 min then cooled down to 0°C. then brine (460 mL) was added. The suspension was stirred for 10 minthen the product was isolated by filtration. The filter cake was washedwith H₂O (300 mL), which was then carefully removed, then dissolved inDCM (300 mL). The solution was dried over MgSO4 then concentrated todryness in vacuo. The crude product was subjected to flashchromatography over silica gel (eluents: DCM and DCM/iPrOH (8:2)) toafford Intermediate 32d-Batch1 (14.4 g, 6.6 mmol).

The same procedure was repeated with Intermediate 32c-Batch1 (23.4 g,9.37 mmol), excluding the flash chromatography, affording Intermediate32d-Batch2 (28.0 g, 9.37 mmol).

Preparation of Intermediate 32e

(Protecting Group Removal)

Intermediate 32d-Batch2 (28.0 g, 9.37 mmol) was dissolved inTFA/DCM/EDT/TIS (90:5:2.5:2.5) (290 mL) and the reaction stirred at rtfor 2 h.

The cleavage solution was filtered off and poured onto cold TBME (3 L)(0-4° C.). The turbid suspension was stirred in an ice-water bath for 30min then filtered through a pore 4 glass filter. The white solid thusobtained was washed with TBME (2×100 mL) then dried in vacuum at 35° C.overnight to afford Intermediate 32e-Batch1 (8.9 g, 5.9 mmol).

The same procedure was repeated with Intermediate 32d-Batch1 (14.4 g,6.6 mmol) yielding Intermediate 32e-Batch2 (9.6 g, 6.3 mmol).

Preparation of Example 32 1) Cyclization

Intermediate 32e (5.0 g, 3.3 mmol) was dissolved in water (500 mL). Asolution of iodine (1.18 g, 4.66 mmol, 1.41 eq) in acetic acid (93 mL)was added in one portion. The reaction mixture was stirred at rt for 10min. A solution of ascorbic acid (1.03 g, 5.83 mmol, 1.77 eq) in water(5.8 mL) was added and the reaction mixture stirred for 10 min, filteredand stored at 4° C. until purification.

The same cyclization procedure was repeated until 18.3 g (12.1 mmol) ofIntermediate 32e had been processed.

2) Purification

The solutions of cyclic peptide were subjected to preparative HPLC inportions of 0.5-5.0 g peptide per injection. The fractions having purityhigher than 95% were pooled and freeze dried to yield a total amount of4.89 g (3.2 mmol) of purified peptide (TFA salt) was produced.

3) Acetate Formation by Ion Exchange

75 g (100 mL) of a strong anion exchanger resin (Ion exchanger III,Merck) in its OH— form was placed in sintered glass filter (porosity 3)and then a solution of acetic acid/water (1:3) (300 mL) was added, thesuspension was manually stirred for 2 min then the resin was drained.The process was repeated with another portion of acetic acid/water (1:3)(300 mL). The resin was washed with deionized water until a neutraldrain was observed. Then the resin was transferred to a 4×20 cm columnequipped with a sintered glass filter (porosity 3).

4.8 g of purified peptide was dissolved in deionized water (50 mL) andadded to the column. The product was eluted with deionized water (200mL). Control of product elution was done by TLC spotting, the richfractions were pooled and freeze dried to give Example 32 (4.1 g, 2.9mmol).

The pure product was analyzed by analytical HPLC (Analytical method B;t_(R)=8.01 min) and UPLC-HRMS (Analytical method D; measured:[M+2H]²⁺=643.328; calculated: [M+2H]²⁺=643.324). The acetate content was7.99-8.27% and the water content was 1.94-1.96%.

The other examples were synthesized in analogy:

-   -   Examples 1 to 8, 10, 11, 13 to 17, 20, 28 to 31 were synthesized        in analogy to Example 12.    -   Examples 19, 26, 27, 36-38 were synthesized in analogy to        Example 18.    -   Examples 22-25, 33-35 were synthesized in analogy to Example 21.

Bioconjugate Examples Example 39:Albumin-PPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-C*-L-S-C*-K-G-P-(D-Nle)Phenethylamine(SEQ ID NO: 62) wherein PPA is 3-(pyridin-2-yldisulfanyl)propanoic acidStep 1: Albumin Decapping

Decapping with TCEP

To a solution of albumin (500 mg, Aldrich, lyophilized powder, fromhuman serum) in 10 mL of PBS 1× buffer in a 15 mL tube was added asolution of TCEP hydrochloride (1.074 mg in bio-grade purified water)once. The resultant solution was shaked at rt for 1 hr, then desaltedand washed with two Amicon Ultra-4 centrifugal filters (30K MWCO). Thefilters were spinned at 4K g for 40 mins and the filtrates werediscarded. 3 mL of bio-grade purified water was added to each filter foreach wash (spinned at 14K g for 10 mins) and the wash process wasrepeated 3 times. The decapped HSA was dissolved in water (−20 mL intotal). The solution was transferred to a 50 mL Falcon tube, andlyophilized to give a crystalline powder (500 mg).

The pure product was analyzed by UPLC-MS (Analytical method F; measured:66439.0; expected: 66437).

Determination of the Number of Free Thiol Group in Decapped HSA

To a solution of this decapped HSA (2 mg) in 400 μL of PBS pH 7.4 in a 2mL tube was added a solution of 6-maleimidohexanoic acid (13 μg) inwater. The resultant solution was shaked at rt for 2 hr. UPLC-MS(Analytical method G) showed mono-adduct formation only, measured:66649.0; expected: 66648.

Decapping with DTT

To a solution of albumin (400 mg, Aldrich, lyophilized powder, fromhuman serum) in 5 mL of PBS 1× buffer in a 15 mL tube was added asolution of DTT (0.232 μl, 2 mg/mL in bio-grade purified water) once.The resultant solution was shaked at rt for 2 hr, then desalted andwashed with twenty Amicon Ultra-0.5 centrifugal filters (10K MWCO). Thefilters were spinned at 14K g for 10 mins and the filtrates werediscarded. Bio-grade purified water was added to the top of each filterfor each wash (spinned at 14K g for 10 mins) and the wash process wasrepeated 6 times. The decapped HSA was dissolved in water (˜20 mL intotal). The solution was transferred to a 50 mL Falcon tube, andlyophilized to give a crystalline powder (376 mg).

The pure product was analyzed by UPLC-MS (Analytical method G; measured:66438.5; expected: 66437).

Determination of the Number of Free Thiol Group in Decapped HSA

To a solution of this decapped HSA (3 mg) in 400 μL of PBS pH 7.4 in a 2mL tube was added a solution of 3-maleimidopropionic acid (25 μg) inwater. The resultant solution was shaked at rt overnight. UPLC-MS(Analytical method G) showed mono-adduct formation only, measured:66608.0; expected: 66606.

Decapping with Cysteine

To a solution of albumin (120 mg, Aldrich, lyophilized powder, fromhuman serum) in 1 mL of 50 mM PBS buffer pH 8.0 in a 2 mL tube was addedcysteine (10.94 mg) once. The resultant solution was shaked at rt for 1hr, then desalted and washed with two Amicon Ultra-0.5 centrifugalfilters (10K MWCO). The filters were spinned at 14K g for 10 mins andthe filtrates were discarded. Bio-grade purified water was added to thetop of each filter for each wash (spinned at 14K g for 10 min) and thewash process was repeated 5 times. The decapped HSA was dissolved inwater (4 mL in total). The solution was transferred to a 15 mL Falcontube, and lyophilized to give a crystalline powder (108 mg).

The pure product was analyzed by UPLC-MS (Analytical method G; measured:66439; expected: 66437).

Determination of the Number of Free Thiol Group in Decapped HSA

To a solution of this decapped HSA (3 mg) in 500 μL of PBS pH 7.4 in a 2mL tube was added a solution of 3-maleimidopropionic acid (15 μg) inwater. The resultant solution was shaked at rt for 1 hr. UPLC-MS(Analytical method G) showed mono-adduct formation only, measured:66608.0; expected: 66606.

Step 2 Synthesis of Peptide-Linker Construct 1:PPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-C*-L-S-C*-K-G-P-(D-Nle)Phenethylamine (SEQID NO: 62) wherein PPA is 3-(pyridin-2-yldisulfanyl)propanoic acid

(SEQ ID NOS 63, 62, 62, and 62, respectively, in order of appearance)

Preparation of Intermediate 1a

(Assembly of Linear Peptide)O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-C*-L-S-C*-K-G-P-(D-Nle)Phenethylamine (SEQ IDNO: 62)

-   -   Phenethylamine-AMEBA resin (Aldrich, 0.25 mmol) is subjected to        solid phase peptide synthesis on the Liberty™ microwave peptide        synthesizer. Coupling is performed as follows:

Number of couplings × Temperature Microwave Coupling AA Reaction time °C. power 1 D-Nle 1 × 7.5 min 50 20 2 P 1 × 7.5 min 50 20 3 G 1 × 7.5 min50 20 4 K(tBoc) 1 × 7.5 min 50 20 5 C(Trt)  1 × 2 min 50 0  1 × 4 min 5025 6 S(tBu) 1 × 7.5 min 50 20 7 L 1 × 7.5 min 50 25 8 C(Trt)  1 × 2 min50 0  1 × 4 min 50 25 9 P 1 × 7.5 min 50 25 10 R(Pbf)  2 × 42 min 50 0 2× 7.5 min 50 25 11 Q(Trt) 1 × 7.5 min 50 25 12 O2Oc 1 × 7.5 min 50 25 13O2Oc 1 × 7.5 min 50 25 14 O2Oc 1 × 7.5 min 50 25 15 O2Oc 1 × 7.5 min 5025 16 TPA(Trt) 1 × 7.5 min 50 25

Preparation of Intermediate 1b

(Cleavage from the Resin with Concomitant Protecting Group Removal thenPurification)

A solution made of 1.54 g of DTT and 0.75 mL of thioanisole in 6 mL ofTFA/TIPS/Water (95:2.5:2.5) is added to Intermediate 1a (0.25 mmol) andthe suspension is shaken at rt for 5 hr. The cleavage solution isfiltered off and the resin is washed with 95% aq. TFA. The combinedcleavage and washing solutions are poured onto cold diethyl ether,giving a precipitate. The suspension is centrifuged and the supernatantpoured off. Diethyl ether is added to the residue, the suspension isvortexed for 3 min, centrifuged, and the supernatant is poured off. Thewashing process is repeated 3 times. The solid is dried in high vacuum.The crude is purified by preparative HPLC and lyophilized from ACN/H₂Oto afford Intermediate 1b.

Preparation of Intermediate 1c

(Cyclization and Purification

Intermediate 1b is dissolved in H₂O (2.0 mL). A solution of 50 mM I₂ inAcOH (1.3 eq) is added in one portion to the stirred solution and thesolution is stirred overnight at rt and LC/MS shows completion of thereaction. 0.5 M Ascorbic acid in H₂O is added to quench the excess ofI₂. The crude is purified by preparative HPLC and lyophilized fromACN/H₂O to afford Intermediate 1c.

Preparation of Peptide-Linker Construct 1 Intermediate 1c

A mixture of Intermediate 1c, 2,2′-dithiodipyridine (3 eq) in ACN isshaked at 25° C. for 1 hr. The reaction mixture is diluted with MeOH andfiltered. The solution is purified by preparative HPLC and lyophilizedfrom ACN/H₂O to afford Peptide-Linker Construct 1.

Step 3: Peptide-Linker Construct/Albumin Conjugation

A solution of decapped HSA (100 mg) in PBS buffer (6 mL) is treated witha solution ofPPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-C*-L-S-C*-K-G-P-(D-N/e)Phenethylamine (SEQID NO: 62) (2 eq in water). The resultant solution is shaked at rt for 1hr, then desalted and washed with 4 Amicon Ultra-0.5 centrifugal filters(10K MWCO). The filters are spinned at 13K g for 10 mins and thefiltrates are discarded. Bio-grade purified water is added to the top ofeach filter for each wash (spinned at 13K g for 10 mins) and the washprocess is repeated 6 times. The conjugate is dissolved in water (4 mLin total). The solution is transferred to a 15 mL Falcon tube, andlyophilized to give Example 39.

Example 40: Apelin Cyclic Peptide Conjugated to a Fatty Acid Via aBCN-PEG Linker Step 1: Preparation of Apelin Cyclic Peptide-BCNConstruct: Preparation ofpE-R-P-C*-L-S-C*-P-N⁶-[[(1α,8α,9α)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl]-K-G-P-(D-Nle)-NH(Phenethyl)[disulfide C4-C7] (SEQ ID NO: 64)

A mixture of pE-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) triacetate[disulfide C4-C7](SEQ ID NO: 50) (Example 32: 100 mg, 0.068 mmol),sodium bicarbonate (38 mg, 0.452 mmol) and water (83 uL) in DMF (1 mL)was stirred at RT for 10 mins, then(1R,8S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succinimidyl carbonate (Berry&associates, 20 mg, 0.068 mmol) was added. The reaction mixture wasstirred at RT for 90 mins. 1 mL of water was added to the mixture, andthe resultant solution was lyophilized to give a powder which was usedfor the next step without further purification.

Step 2: Di-tert-butyl 2-(undec-10-yn-1-yl)malonate

Di-tert-butyl malonate (800 mg, 3.70 mmol) is dissolved in DMF (9 mL) at0° C. under N₂ and NaH (148 mg, 3.70 mmol) is added. The reaction isstirred 30 minutes at 0° C. and 11-bromo-dec-1-ene (3.33 mmol) is addedslowly dropwise, resulting in a yellow solution. The reaction is stirredat 0° C. for 2 hours then warmed to r.t. and stirred for 16 hours. Themixture is taken up in EtOAc (75 mL) and washed with H₂O (25 mL). Theaqueous layer is extracted with EtOAc (75 mL) and the combined organiclayers are dried over Na₂SO₄, filtered and concentrated. The mixture ispurified via flash column (12 g silica cartridge, 0-20% EtOAc/heptanes)and fractions are concentrated to yield the desired product.

Step 3: 11,11-di-tert-butyl 1-ethyl docos-21-ene-1,11,11-tricarboxylate

Compound from step 2 (0.442 mmol) is dissolved in DMF (2 mL) at 0° C.and NaH (21.23 mg, 0.531 mmol) is added. The reaction stirred at 0° C.for 15 minutes and ethyl 11-bromoundecanoate (143 mg, 0.486 mmol) isadded slowly dropwise. The reaction is warmed to r.t. and stirred for 16hours. The mixture is diluted with EtOAc (40 mL) and washed once withH₂O (20 mL). The aqueous layer is extracted once with EtOAc (40 mL) andthe organic layers are combined, dried over Na₂SO₄, filtered andconcentrated. The sample is dissolved in 1 mL DCM and purified via flashcolumn (12 g silica column, 0-20% EtOAc/heptane, 15 min). The fractionsare combined and concentrated to give the desired product.

Step 4: 12,12-bis(tert-butoxycarbonyl)tricos-22-enoic acid

To a solution of compound from step 3 (0.037 mmol) in ^(t)BuOH (1 mL) isadded a solution of KOtBu (114 mg, 1.012 mmol) in ^(t)BuOH (2 mL) at 30°C. under N₂. The mixture is stirred at r.t. and monitored by TLC (1:1EtOAc/hexanes, KMnO₄, reflux). After the reaction is completed, thereaction mixture is quenched with 1 M HCl (20 mL) and extracted twicewith EtOAc (25 mL). The organic layers are combined, dried over Na₂SO₄,filtered and concentrated. The material was carried on to the next stepwithout further purification.

Step 5: Docos-21-ene-1,11,11-tricarboxylic acid

TFA (2 mL) is added to a compound from step 4 (0.022 mmol) and thereaction is stirred at r.t. for 1 hour. The mixture is diluted with DCM(10 mL) and concentrated. The material is taken up in EtOAc (10 mL) andwashed with H₂O (20 mL). The organic layer is dried over Na₂SO₄,filtered and concentrated. The crude material is dissolved in 1 mL MeOHand purified via MS-triggered HPLC (Sunfire 30×50 mm 5 um column ACN/H₂Ow/0.1% TFA 75 ml/min, 1.5 ml injection, 45-70% ACN over 3.5 min).

Step 6:2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-(undec-10-en-1-yl)tridecanedioicacid

DCC (187 mg, 0.908 mmol) in DCM (2 mL) was added to a solution ofN-hydroxysuccinimide (99 mg, 0.862 mmol) anddocos-21-ene-1,11,11-tricarboxylic acid (Intermediate 45: 400 mg, 0.908mmol) in DCM (7 mL) and THF (0.7 mL). The reaction was stirred overnightbefore the solvent was evaporated. The residue was purified by HPLC(Sunfire C18 30×50 mm; 55-80% ACN/water+0.1% TFA) to yield the titlecompound (155 mg, 0.288 mmol, 32%): by LCMS Method E Rt=1.51 min, M+H538.3; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.16-1.46 (m, 28H) 1.60-1.87(m, 3H) 1.91-2.17 (m, 5H) 2.38 (t, J=7.03 Hz, 2H) 2.86 (br. s., 4H) 3.68(dd, J=11.25, 7.34 Hz, 1H) 3.78 (dd, J=11.31, 5.20 Hz, 1H) 3.99-4.10 (m,1H).

Step 7: Fatty Acid-PEG Linker

Azido-dPEG23-amine (Quanta Biodesign: 164 mg, 0.149 mmol) and compoundfrom step 6 (80 mg, 0.149 mmol) were dissolved in THF (2.5 mL). DIPEA(39 μL, 0.233 mmol) was added and the reaction agitated overnight. Thesolvent was evaporated and the residue purified by HPLC (Sunfire C1830×50 mm; 45-70% ACN/water+0.1% TFA) to yield compounds A (97 mg, 0.061mmol, 41%) and B (32 mg, 0.021 mmol, 14%): LCMS ZQ1 Method E Rt=1.35min, [M+2H]⁺² 761.9; ¹H NMR (400 MHz, ACETONITRILE-d3) δ ppm 1.05-1.18(m, 3H) 1.19-1.32 (m, 20H) 1.36 (t, J=7.15 Hz, 1H) 1.48-1.59 (m, 2H)1.65-1.75 (m, 2H) 2.01-2.06 (m, 2H) 2.25 (t, J=7.46 Hz, 2H) 3.33-3.39(m, 2H) 3.39-3.44 (m, 2H) 3.50-3.67 (m, 98H) 4.84-4.95 (m, 1H) 4.95-5.06(m, 1H) 5.83 (ddt, J=17.07, 10.29, 6.68, 6.68 Hz, 1H) 7.31 (t, J=5.44Hz, 1H); LCMS method E Rt=1.50 min, [M+2H]⁺² 739.9; ¹H NMR (400 MHz,ACETONITRILE-d3) □ ppm 1.16-1.42 (m, 30H) 1.42-1.63 (m, 5H) 2.00-2.07(m, 2H) 2.22-2.28 (m, 2H) 2.40-2.52 (m, 2H) 3.25-3.33 (m, 2H) 3.33-3.42(m, 2H) 3.42-3.50 (m, 2H) 3.50-3.68 (m, 88H) 4.86-5.06 (m, 2H) 5.83(ddt, J=17.04, 10.26, 6.71, 6.71 Hz, 1H) 6.40-6.74 (m, 1H).

Step 8: Preparation of Example 40

A mixture ofpE-R-P-C*-L-S-C*-N-[[(1α,8α,9α)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl]-K-G-P-(D-Nle)-NH(Phenethyl)[disulfide C4-C7] (SEQ ID NO: 64) (50 mg of the product from step 1 in 1mL of water, 0.034 mmol) and compound A from step 7 (52 mg, in 268 uL ofwater) was stirred at RT for about 3 hrs. The reaction mixture was thenpurified by preparative HPLC (Sunfire 30×50 mm 5 um column ACN/H2Ow/0.1% TFA 75 ml/min, 15-40% ACN 5 min gradient). The product fractionwas lyophilized to give the titled product as TFA salt (24 mg, 21%).LCMS (Waters Acquity UPLC BEH C18 1.7 um 2.1×50 mm, 50° C., Eluent A:Water+0.1% Formic Acid, Eluent B: Acetonitrile+0.1% Formic Acid,gradient 2% to 98% B/A over 5.15 mins): Rentention time: 2.77 mins; MS[M+2]²⁺: observed: 1491.8808, calculated: 1491.8560.

Example 41: Apelin Cyclic Peptide Conjugate to a Fatty Acid at theN-Terminus Step 1: Synthesis of A-H-Q-R-P-C-L-S-C-K-G-P-DNle-PhenethylAmine Intermediate (SEQ ID NO: 65) 41a

Phenethylamine-AMEBA resin (Sigma Aldrich, 0.1 mmol, 1.0 mmol/g) wassubjected to solid phase peptide synthesis on an automatic peptidesynthesizer (CEM Liberty Blue Microwave) with standard double Arg forthe Arg residues and DNle coupled double time. Amino acids were preparedas 0.2 M solutions in DMF. A standard coupling cycle was defined asfollows:

-   -   Amino acid coupling: AA (5 eq.), HATU (5 eq.), DIEA (25 eq.)    -   Washing: DMF (3×7 mL)    -   Fmoc Deprotection: 20% Piperidine/0.1 M HOBt (2×7 mL)    -   Washing: DMF (4×7 mL then 1×5 mL)

Cou- Number of couplings × Coupling pling AA Reaction time (Temp) Method1 Fmoc-D-Nle-OH 1 × 10 min (70° C.)  DIEA/HATU 2 Fmoc-L-Pro-OH 1 × 5 min(70° C.) DIEA/HATU 3 Fmoc-L-Gly-OH 1 × 5 min (70° C.) DIEA/HATU 4Fmoc-L-Lys-OH 1 × 5 min (70° C.) DIEA/HATU 5 Fmoc-L-Cys-OH 1 × 5 min(70° C.) DIEA/HATU 6 Fmoc-L-Ser-OH 1 × 5 min (70° C.) DIEA/HATU 7Fmoc-L-Leu-OH 1 × 5 min (70° C.) DIEA/HATU 8 Fmoc-L-Cys-OH 1 × 5 min(70° C.) DIEA/HATU 9 Fmoc-L-Pro-OH 1 × 5 min (70° C.) DIEA/HATU 10Fmoc-L-Arg-OH 2 × 25 min (25° C.)  DIEA/HATU 11 Fmoc-L-Gln-OH 1 × 5 min(70° C.) DIEA/HATU 12 Fmoc-L-His-OH 1 × 5 min (70° C.) DIEA/HATU 13Fmoc-L-Ala-OH 1 × 5 min (70° C.) DIEA/HATU

After the assembly of the peptide, the resin was washed with DMF (2×50mL) and DCM (2×50 mL) then dried under vacuum to give Intermediate 41a(276 mg, 0.1 mmol).

Step 2: Preparation of Intermediate 41 b (Cleavage of Peptide fromResin)

Intermediate 41a (276 mg, 0.1 mmol) was combined with 4 mL TFA solution(37 mL TFA, 1 mL H₂O, 1 mL TIPS, 3.06 g DTT) and shaken at r.t. for 3hours. The solution was removed from the resin and precipitated into 40mL cold Et₂O. The solution was vortexed and let stand over ice for 10minutes before centrifuging at 4000 rpm for 5 minutes. The solvent wasremoved and the white solid was washed twice more with cold Et₂O (40 mLeach time), centrifuged (5 minutes each time) and decanted. The solidwas dried under vacuum overnight yielding Intermediate 41b-batch 1 (17.4mg, 0.012 mmol). LCMS (SQ2 ProductAnalysis-Acidic-Peptide-Polar, AcquityUPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=1.83minutes, MS [M+H] 1513.5.

Step 3: Preparation of Intermediate 41c (Cyclization of CysteineResidues)

Intermediate 41b-batch1 and 2 (29.6 mg, 0.020 mmol) was dissolved inwater (3 mL) and 10 drops of DMSO to give a slightly cloudy solution.Iodine (50 mM in HOAc, 0.783 mL, 0.039 mmol) was added slowly dropwiseand the reaction was mixed at r.t. overnight. LCMS analysis of the crudereaction showed complete conversion of starting material. 0.5 M ascorbicacid was added dropwise until color dissipated. The material waspurified via MS-triggered HPLC. Lyophilization of the pooled fractionsgave 7 mg of the desired product as a white powder (4.63 μmol, 24%).LCMS (SQ2 ProductAnalysis-Acidic-Peptide, Acquity UPLC BEH C18 column,130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=0.90 minutes, MS [M+H]1511.8.

Step 4: 1-Benzyl 3-tert-butyl 2-undecylmalonate

To a suspension of NaH (160 mg, 4.0 mmol) in DMF (8 mL) at 0° C. underN₂, was added benzyl tert-butyl malonate (1.0 g, 4.0 mmol) in DMF (2mL). The mixture was stirred for 50 min after which 1-bromoundecane inDMF (2 mL) was added. After an additional hour of stirring the reactionwas allowed to warm to room temperature. The reaction was maintainedovernight. Et₂O (100 mL) and water (20 mL) were added to partition thereaction. The aqueous phase was extracted with Et₂O (100 mL), and thecombined organics dried over Na₂SO₄. The solvent was evaporated and theresidue purified by flash column (C18 12 g, 40-100% ACN/water+0.1% TFA)to yield the title compound as a colorless oil (1.14 g, 2.82 mmol, 71%):LCMS Method F Rt=1.58 min, M+Na 427.4; ¹H NMR (400 MHz, CHLOROFORM-d) δppm 0.84-0.96 (m, 3H) 1.28 (br. s, 12H) 1.31 (m, J=3.90 Hz, 6H) 1.41 (s,9H) 1.88 (q, J=7.38 Hz, 2H) 3.29 (t, J=7.58 Hz, 1H) 5.19 (q, J=12.27 Hz,2H) 7.30-7.42 (m, 5H).

Step 5: 1,11-Dibenzyl 11-tert-butyl docosane-1,11,11-tricarboxylate

The title compound was synthesized in a fashion similar to step 3 ofExample 40 using compound from step 4 (177 mg, 0.284 mmol) as a startingmaterial to yield a colorless oil (153 mg, 0.213 mmol, 75%): ¹H NMR (400MHz, CHLOROFORM-d) δ ppm 0.86-0.93 (m, 3H) 1.12-1.21 (m, 2H) 1.21-1.37(m, 30H) 1.66 (quin, J=7.40 Hz, 2H) 1.89-2.07 (m, 4H) 2.37 (t, J=7.58Hz, 2H) 2.84 (br. s., 4H) 5.13 (s, 2H) 5.25 (s, 2H) 7.30-7.47 (m, 10H).

Step 6:13-(Benzyloxy)-2-((benzyloxy)carbonyl)-13-oxo-2-undecyltridecanoic acid

To a solution of a compound from step 5 (200 mg, 0.295 mmol) in DCM (3mL) was added TFA (0.6 mL), and the reaction stirred at room temperaturefor 3 hrs. The solvent was evaporated and the residue purified by flashcolumn (silica 12 g, 0-15% EtOAc/HEP) to yield the title compound (177mg, 0.284 mmol, 96%): ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.87-0.94 (m,3H) 0.94-1.05 (m, 2H) 1.19 (br. s., 14H) 1.23-1.37 (m, 16H) 1.65 (quin,J=7.40 Hz, 2H) 1.78-1.91 (m, 2H) 1.93-2.05 (m, 2H) 2.37 (t, J=7.52 Hz,2H) 5.14 (s, 2H) 5.27 (s, 2H) 7.31-7.44 (m, 10H).

Step 7: 1,11-Dibenzyl 11-(2,5-dioxocyclopentyl)docosane-1,11,11-tricarboxylate

The title compound was synthesized in a fashion similar to step 6 ofExample 40 using compound from step 6 (177 mg, 0.284 mmol) as a startingmaterial to yield a colorless oil (153 mg, 0.213 mmol, 75%): ¹H NMR (400MHz, CHLOROFORM-d) δ ppm 0.86-0.93 (m, 3H) 1.12-1.21 (m, 2H) 1.21-1.37(m, 30H) 1.66 (quin, J=7.40 Hz, 2H) 1.89-2.07 (m, 4H) 2.37 (t, J=7.58Hz, 2H) 2.84 (br. s., 4H) 5.13 (s, 2H) 5.25 (s, 2H) 7.30-7.47 (m, 10H).

Step 8

A solution of intermediate 34 (145 mg, 0.201 mmol) in THF (1.5 mL) andDCM (1.5 mL) was added to a vial charged with amino-PEG24-acid. DIPEA(88 μL, 0.504 mmol) was added and the reaction agitated on a shakerplate for 15 hrs. The solvent was evaporated and the residue purified bysupercritical fluid chromatography (Waters HILIC 20×150 mm; 15-25%MeOH/CO₂) to yield intermediate 35 (151 mg, 0.086 mmol, 43%): LCMSMethod E Rt=1.30 min, [M+2H]+2 876.4; ¹H NMR (400 MHz, CHLOROFORM-d) δppm 0.86-0.93 (m, 3H) 0.93-1.04 (m, 2H) 1.19 (br. s., 15H) 1.23-1.37 (m,15H) 1.61-1.68 (m, 2H) 1.78 (td, J=12.44, 4.34 Hz, 2H) 1.92-2.05 (m, 2H)2.37 (t, J=7.58 Hz, 2H) 2.62 (t, J=6.05 Hz, 2H) 3.49 (dd, J=6.72, 2.32Hz, 2H) 3.52-3.59 (m, 2H) 3.59-3.73 (m, 92H) 3.80 (t, J=6.05 Hz, 2H)5.13 (s, 2H) 5.18 (s, 2H) 7.31-7.42 (m, 10H) 8.09 (t, J=5.26 Hz, 1H).

Step 9

DCC (22 mg, 0.103 mmol) in DCM (0.265 mL) was added to a solution ofintermediate 35 (150 mg, 0.086 mmol) and N-hydroxysuccinimide in DCM(1.5 mL). The reaction was stirred for 1.5 hrs. AdditionalN-hydroxysuccinimide (10 mg) in THF (0.5 mL) and DCC (22 mg) in DCM(0.265 mL) was added and the reaction stirred overnight. The solvent wasevaporated and the residue purified by flash column (silica 12, 0-5%MeOH/DCM) to yield intermediate 36 (159 mg, quantitative) as a whitesolid: LCMS Method G Rt=1.55 min, [M+H₃O+H]⁺² 933.9

Step 10

To a solution of compound from step 9 (159 mg, 0.086 mmol) in THF (5 mL)was added a suspension of 10% Pd on carbon (4.6 mg, 4.3□mol) in THF (1mL). The reaction was placed under hydrogen and stirred for 40 min. MorePd on carbon (7 mg, 6.5 μmol) was added and the stirred another 1 hrunder hydrogen. The reaction was passed through a membrane filter andthe filtrate evaporated. The residue was purified by HPLC (Sunfire C1830×50 mm, 45-70% ACN/water+0.1% TFA) to yield the title compound (83 mg,0.047 mmol, 54%): LCMS Method G Rt=1.03 min, [M+2H]+2 835.2; ¹H NMR (400MHz, CHLOROFORM-d) □ ppm 0.84-0.94 (m, 3H) 1.17 (br. s., 2H) 1.21-1.39(m, 30H) 1.57-1.68 (m, 2H) 1.69-1.80 (m, 2H) 1.97-2.10 (m, 2H) 2.34 (t,J=7.21 Hz, 2H) 2.86 (s, 4H) 2.92 (t, J=6.48 Hz, 2H) 3.51-3.73 (m, 96H)3.87 (t, J=6.48 Hz, 2H) 7.45 (t, J=4.46 Hz, 1H)

Step 4: Preparation of Conjugate Comprising Apelin ConstructA-H-Q-R-P-C-L-S-C-K-G-P-DNle-Phenethyl Amine (SEQ ID NO: 65) and FattyAcid-PEG Construct (N-Terminus Conjugation)—Example 41

A 10 mg/mL solution of NHS-fatty acid (product A from step 7 of Example40) was prepared in H₂O. Intermediate 41c (1.5 mg, 0.993 μmol) wasdissolved in 30 mM pH4 NaOAc buffer (672 μL) and NHS-fatty acid (0.850mL, 5.10 μmol) was added. The reaction was mixed at r.t. for 16 hours atwhich point an additional 1.5 mg of NHS-fatty acid (10 mg/mL in H₂O) wasadded and the reaction mixed at r.t. for 16 hours. 8 mg of NHS-fattyacid (10 mg/mL in H₂O) was added and the reaction mixed at r.t. for 3days and 1.7 mg of NHS-fatty acid (10 mg/mL in H₂O) was added. Themixture was shaken at r.t. for 16 hours and purified via M-triggeredHPLC to give 1.7 mg of the title compound as a white powder (0.510 μmol,51%). LCMS (SQ2 ProductAnalysis-Acidic-Peptide-Polar, Acquity UPLC BEHC18 column, 130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=3.87 minutes, MS[M+H+2/2] 1533.1; [M+H+3/3] 1022.9.

Example 42: Conjugation of AH-Fc with Apelin Cyclic Peptide GeneralScheme

Step 1: Preparation of AH-Fc Construct Construct Cloning:

A DNA fragment containing the mouse Ig kappa chain signal peptidefollowed by a human Fc and a short bis-amino acid sequence (AH) wascodon optimized by gene synthesis (GeneArt) with 5′-NheI and 3′-EcoRIrestriction sites. The resulting sequence was restriction digested withboth NheI and EcoRI and ligated into NheI and EcoRI sites of vectorpPL1146, downstream of a CMV promoter. The ligation was transformed intoE coli DH5α cells and colonies containing the correct insert wereidentified by DNA sequencing. Sequence shown is for the sense strand andruns in the 5′ and 3′ direction.

AH-Fc (SEQ ID NO: 66) GCTAGCCACCATGGAAACTGACACCCTGCTGCTGTGGGTCCTGCTGCTGTGGGTGCCTGGCAGCACTGGCGCTCATGATAAGACACACACATGCCCCCCTTGTCCAGCACCAGAGGCAGCTGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACACTGATGATCTCAAGGACCCCAGAAGTCACATGCGTGGTCGTGGACGTGTCTCACGAGGACCCCGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCTAAGACCAAACCCCGAGAGGAACAGTACAACAGCACCTATCGGGTCGTGTCCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTATAAGTGCAAAGTGAGTAATAAGGCTCTGCCTGCACCAATCGAGAAAACAATTTCTAAGGCTAAAGGGCAGCCAAGAGAACCCCAGGTGTACACTCTGCCTCCATCTAGGGAGGAAATGACAAAGAACCAGGTCAGTCTGACTTGTCTGGTGAAAGGCTTCTACCCCTCCGACATCGCAGTGGAGTGGGAATCTAATGGCCAGCCTGAAAACAATTACAAGACCACACCCCCTGTGCTGGACTCCGATGGGTCTTTCTTTCTGTATTCTAAGCTGACCGTGGATAAAAGTCGGTGGCAGCAGGGAAACGTCTTCTCATGCAGCGTGATGCACGAGGCCCTGCACAATCATTACACACAGAAGTCCCTGTCTCTGAGTCC AGGCAAATGAGAATTCSequence of the AH-Fc construct: (SEQ ID NO: 67) 1METDTLLLWV LLLWVPGSTG AHDKTHTCPP CPAPEAAGGP SVFLFPPKPK 51DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 101TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 151YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 201DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

AH-Fc Protein Expression and Purification:

AH-Fc expression plasmid DNA was transfected into HEK293T cells at adensity of 1×10⁶ cells per ml using standard polyethylenimine methods.500 ml cultures were then grown in FreeStyle 293 Medium (LifeTechnologies) in 3 L flasks for 4 days at 37° C.

AH-Fc protein was purified from clarified conditioned media. Briefly 500ml of conditioned media was flowed over a 5 ml HiTrap MabSelect SuRecolumn (GE Life Sciences) at 4 ml/min. The column was washed with 20column volumes of PBS containing 0.1% Triton X-114 and then the AH-Fcprotein was eluted with 0.1M glycine, pH 2.7, neutralized with 1 MTris-HCl, pH 9 and dialyzed against PBS. Protein yields were 10 to 20 mgper 500 ml conditioned media and endotoxin levels were <1 EU/mg asmeasured by the Charles River ENDOSAFE PTS test.

Quality Control of AH-Fc Proteins: LC/MS of AH-Fc Proteins:

Peaks were heterogeneous and about 3 kDa larger than expected fordimers. This is characteristic of N-linked glycosylation expected for Fcwhich has a consensus N-linked glycosylation site.

LC/MS of Reduced, N-Deglycosylated AH-Fc Proteins:

gave sharp peaks. The molecular weight for AH-Fc was as expected. Thecysteines at the C-terminus appear to protect the protein from cleavage.

Analytical Size Exclusion on Superdex 200:

Fc-Apelin proteins have between 89 and 100% dimer, 0 to 10% tetramer,and 0 to 1% aggregate.

Reducing SDS/PAGE:

All proteins migrated as predominately monomers of the expected size.

Step 2: Synthesis ofNH₂-AzidoLys-GGGGS-Q-R-P-C-L-S-C-K-G-P-Dnle-Phenethylamine (SEQ ID NO:68)

Phenethylamine-AMEBA resin (Sigma Aldrich, 0.25 mmol, 1.0 mmol/g) wassubjected to solid phase peptide synthesis on an automatic peptidesynthesizer (CEM Liberty Blue Microwave) with standard double Arg forthe Arg residues and D-Nle and Azidolysine coupled double time. Aminoacids were prepared as 0.2 M solution in DMF. A standard coupling cyclewas defined as follows:

-   -   Amino acid coupling: AA (5 eq.), HATU (5 eq.), DIEA (25 eq.)    -   Washing: DMF (3×7 mL)    -   Fmoc Deprotection: 20% Piperidine/0.1 M HOBt (2×7 mL)    -   Washing: DMF (4×7 mL then 1×5 mL)

Cou- Number of couplings × Coupling pling AA Reaction time (Temp) Method1 Fmoc-D-Nle-OH 1 × 10 min (70° C.)  DIEA/HATU 2 Fmoc-L-Pro-OH 1 × 5 min(70° C.) DIEA/HATU 3 Fmoc-L-Gly-OH 1 × 5 min (70° C.) DIEA/HATU 4Fmoc-L-Lys-OH 1 × 5 min (70° C.) DIEA/HATU 5 Fmoc-L-Cys-OH 1 × 5 min(70° C.) DIEA/HATU 6 Fmoc-L-Ser-OH 1 × 5 min (70° C.) DIEA/HATU 7Fmoc-L-Leu-OH 1 × 5 min (70° C.) DIEA/HATU 8 Fmoc-L-Cys-OH 1 × 5 min(70° C.) DIEA/HATU 9 Fmoc-L-Pro-OH 1 × 5 min (70° C.) DIEA/HATU 10Fmoc-L-Arg-OH 2 × 25 min (25° C.)  DIEA/HATU 11 Fmoc-L-Gln-OH 1 × 5 min(70° C.) DIEA/HATU 12 Fmoc-L-Ser-OH 1 × 5 min (70° C.) DIEA/HATU 13Fmoc-L-Gly-OH 1 × 5 min (70° C.) DIEA/HATU 14 Fmoc-L-Gly-Gly- 1 × 10 min(70° C.)  DIEA/HATU Gly-OH 15 Fmoc-L-AzidoLys- 1 × 10 min (70° C.) DIEA/HATU OH

After the assembly of the peptide, the resin was washed with DMF (2×50mL) and DCM (2×50 mL) then dried under vacuum to give Intermediate 42a(770 mg, 0.250 mmol).

Step 3: Preparation of Intermediate 42b (Cleavage of Peptide from Resin)

Intermediate 42a (770 mg, 0.250 mmol) was divided in half and eachsample was combined with 6 mL TFA solution (37 mL TFA, 1 mL H₂O, 1 mLTIPS, 2.569 g (20 eq.) DTT) and shaken at r.t. for 3 hours. The solutionwas removed from the resin and precipitated into 40 mL cold Et₂O. Thesolution was vortexed and let stand over ice for 10 minutes beforecentrifuging at 4000 rpm for 5 minutes. The solvent was removed and thewhite solid was washed twice more with cold Et₂O (40 mL each time),centrifuged (5 minutes each time) and decanted. The solid was driedunder vacuum overnight and purified via M-triggered HPLC yieldingIntermediate 43b as a white powder (80 mg, 0.045 mmol, 80%). LCMS (SQ2ProductAnalysis-Acidic-Peptide-Polar, Acquity UPLC BEH C18 column, 130Å, 1.7 μm, 2.1 mm×50 mm, 50° C.): R_(t)=2.32 minutes, MS [M+H+2/2]888.0.

Step 4: Preparation of Intermediate 42c (Cyclization of CysteineResidues)

Intermediate 42b (80 mg, 0.045 mmol) was dissolved in water (1.25 mL)and iodine (50 mM in HOAc, 1.804 mL, 0.090 mmol) was added slowlydropwise and the reaction was mixed at r.t. for 3 hours. LCMS analysisof the crude reaction showed complete conversion of starting material.0.5 M ascorbic acid was added dropwise until color dissipated. Thematerial was purified via MS-triggered HPLC. Lyophilization of thepooled fractions gave 20.1 mg of the desired product as a white powder(0.045 mmol, 25%). LCMS (SQ2 ProductAnalysis-Acidic-Peptide-Polar,Acquity UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm×50 mm, 50° C.):R_(t)=2.17 minutes, MS [M+H+2/2] 886.8.

Step 5: Preparation of Fc-AH BCN (Installation of Click Handle on AH-FcN-Terminus)

Fc-AH (from step 1: 1 mL, 3 mg/mL, 0.117 μmol) was dissolved in 30 mMNaOAc pH 4.0 (4.3 mL) and a 10 mg/mL stock solution of(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl)carbonate (BCN) in DMSO (0.70 mL) was slowly added and the reactionplaced on a shaker plate at r.t. for 3 days. BCN (0.25 mL) was added andthe reaction was mixed at r.t. for 3 days. BCN (0.70 mL) was added andthe reaction was mixed at r.t. for 16 hours. The solution was exchangedinto 30 mM NaOAc pH 4.0 using 10 kDa MWCO Amicon centrifugal filter bydiluting and concentrating the reaction 5 times to a volume of 900 μL.The solution was centrifuged and supernatant removed. The concentrationwas measured by A280 to be 1.73 mg/mL (1.56 mg, 26%). LCMS (QT2,Protein_20-70 kDa_3 min, Proswift Monolith 4.6×50 mm, 50° C., Eluent A:Water+0.1% Formic Acid, Eluent B: CAN+0.1% Formic Acid, 2-98% over 2min) R_(t)=1.58 min.

% TIC (MS+) Degree of Labelling Calculated Observed Intensity AH-Fc51141 51141.5 18 AH-Fc + 1BCN 51318 51317 31 AH-Fc + 2BCN 51495 51496 31AH-Fc + 3BCN 51672 51671 20

Step 6: Preparation of Example 42: Fc-HA-BCN Conjugated to Intermediate43c

A 50 mg/mL stock solution of intermediate 42c in H₂O was prepared.Intermediate 43c (53.7 μL, 1.515 μmol) was added to a stock solution ofFc-HA-BCN (from step 5) in 30 mM NaOAc pH 4.0 (1.73 mg/mL, 1.56 mg,0.030 μmol) and the reaction was mixed at r.t. for 16 hours. Thesolution was exchanged into 30 mM NaOAc pH 4.0 using 50 kDa MWCO Amiconcentrifugal filter by diluting and concentrating the reaction 5 times toa volume of 250 μL. The concentration was measured by A280 to be 3.32mg/mL (830 μg, 50%). LCMS (QT2, Protein_35-70 kDa_3 min, ProswiftMonolith 4.6×50 mm, 50° C., Eluent A: Water+0.1% Formic Acid, Eluent B:CAN+0.1% Formic Acid, 10-80% B over 2 min) R_(t)=1.43 min, MS[M(glycosylated)+H] 54524.5.

Example 43: Fc-Apelin Conjugate Using Sortase (SEQ ID NOS 69, 69, 70,71, and 71, Respectively, in Order of Appearance)

Step 1: Preparation of Fc-Sortase Construct Construct Cloning:

A DNA fragment containing the mouse Ig kappa chain signal peptidefollowed by a human Fc and a sortase recognition sequence (LPXTG) (SEQID NO: 72) was codon optimized by gene synthesis (GeneArt) with 5′-NheIand 3′-EcoRI restriction sites. The resulting sequence was restrictiondigested with both NheI and EcoRI and ligated into NheI and EcoRI sitesof vector pPL1146, downstream of a CMV promoter. The ligation wastransformed into E coli DH5a cells and colonies containing the correctinsert were identified by DNA sequencing. Sequence shown is for thesense strand and runs in the 5′ and 3′ direction.

Fc-sortase (SEQ ID NO: 73)GCTAGCCACCATGGAAACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCGATAAGACCCACACCTGTCCTCCCTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGAAAAGGCGGCGGAGGCTCTCTGCCTGAAACAGGCGGACTGGAAGTGCTGTTCC AGGGCCCCTAAGAATTC (SEQ ID NO: 74) 1 METDTLLLWV LLLWVPGSTG DKTHTCPPCP APEAAGGPSV FLFPPKPKDT51 LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY 101RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 151LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 201DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG 251GSLPETGGLEVLFQGP

Protein Expression and Purification:

Fc-sortase expression plasmid DNA was transfected into HEK293T cells ata density of 1×10⁶ cells per ml using standard polyethylenimine methods.500 ml cultures were then grown in FreeStyle 293 Medium (LifeTechnologies) in 3 L flasks for 4 days at 37° C.

Fc-sortase protein was purified from clarified conditioned media.Briefly, 500 ml of conditioned media was flowed over a 5 ml HiTrapMabSelect SuRe column (GE Life Sciences) at 4 ml/min. The column waswashed with 20 column volumes of PBS containing 0.1% Triton X-114 andthen the Fc-sortase protein was eluted with 0.1M glycine, pH 2.7,neutralized with 1 M Tris-HCl, pH 9 and dialyzed against PBS. Proteinyields were 10 to 20 mg per 500 ml conditioned media and endotoxinlevels were <1 EU/mg as measured by the Charles River ENDOSAFE PTS test.

Quality Control of Fc-Sortase Protein LC/MS of Native Fc-SortaseProtein:

Peak was heterogeneous and about 3 kDa larger than expected for dimers.This is characteristic of N-linked glycosylation expected for Fc whichhas a consensus N-linked glycosylation site.

LC/MS of Reduced, N-Deglycosylated Fc-Sortase Protein:

Peak was sharp. The molecular weight was 2 daltons less thantheoretical, likely due to Cysteine×2 reduction.

Analytical Size Exclusion on Superdex 200:

Fc-sortase protein had between 89 and 100% dimer, 0 to 10% tetramer, and0 to 1% aggregate.

Reducing SDS/PAGE:

The protein migrated predominately as a monomer of the expected size.

Step 2: Preparation of Apelin Peptide(H₂N-GGGGGQRPC*LSC*KGP(D-Nle)Phenethylamine) (SEQ ID NO: 70) for SortaseConjugation

(SEQ ID NOS 75, 70, and 70, respectively, in order of appearance)

Step 2a: Preparation of Intermediate 43a

Phenethylamine-AMEBA resin (Sigma Aldrih, 0.25 g, 0.25 mmol, 1.0 mmol/g)was subjected to solid phase peptide synthesis on an automatic peptidesynthesizer (CEM LIBERTY) with standard double Arg for the Arg residues.Amino acids were prepared as 0.2 M solutions in DMF.

A coupling cycle was defined as follows:

-   -   Amino acid coupling: AA (4.0 eq.), HATU (4.0 eq.), DIEA (25 eq.)    -   Washing: DMF (3×10 mL, 1 min each time).    -   Fmoc deprotection: Piperidine/DMF (1:4) (10 mL, 75° C. for 1        min, then 10 mL, 75° C. for 3 min).    -   Washing: DMF (4×10 mL, 1 min each time).

Cou- Number of couplings × Reaction pling AA Reaction time Temperature 1Fmoc-D-Nle-OH 1 × 5 min 75° C. 2 Fmoc-L-Pro-OH 1 × 5 min 75° C. 3Fmoc-Gly-OH 1 × 5 min 75° C. 4 Fmoc-L-Lys(Boc)-OH 1 × 5 min 75° C. 5Fmoc-L-Cys(Trt)-OH 1 × 6 min 2 min at 25° C. 4 min at 50° C. 6Fmoc-L-Ser(tBu)-OH 1 × 5 min 75° C. 7 Fmoc-L-Leu-OH 1 × 5 min 75° C. 8Fmoc-L-Cys(Trt)-OH 1 × 6 min 2 min at 25° C. 4 min at 50° C. 9Fmoc-L-Pro-OH 1 × 5 min 75 10 Fmoc-L-Arg(Pbf)-OH 2 × 30 min  25 min at25° C.  5 min at 75° C. 11 Fmoc-L-Gln(Trt)-OH 1 × 5 min 75° C. 12Fmoc-Gly-Gly-Gly- 1 × 5 min 75° C. OH 13 Fmoc-Gly-OH 1 × 5 min 75° C. 14Fmoc-Gly-OH 1 × 5 min 75° C.

After the assembly of the peptide, the resin was washed with DMF (3×10mL), DCM (3×10 mL). The peptide resin was dried under vacuum at roomtemperature to give Intermediate 43a (0.622 g, 0.25 mmol).

Step 2b: Preparation of Intermediate 42b,H₂N-G-G-G-G-G-Q-R-P-C-L-S-C-K-G-P-(D-Nle)-NH(Phenethyl) (SEQ ID NO: 70)

1) Cleavage and Protecting Group Removal

To intermediate 43a (0.622 g, 0.25 mmol) was added 3 mL solution of 95%TFA/2.5% H₂O/2.5% TIPS and DTT (771 mg, 5.00 mmol), the resultingmixture was shaked at room temperature for 3 hours, then filtered. Thefiltrate was dropped into 40 mL of cold ether, then centrifuged at 4000rpm for 5 minutes. The solvent was removed and the white solid waswashed with ether (3×40 mL), vortexed and centrifuged. The solid wasdried under high vacuum at 25° C. for 1 hour.

2) Purification

The above white solid was then purified by preparative HPLC (Sunfire™Prep C18 OBD™ 30×50 mm 5 um column ACN/H₂O w/0.1% TFA 75 ml/min, 10-30%ACN 8 min gradient). The product fraction was lyophilized to giveintermediate 43b as TFA salt (44 mg, 11%).

Step 3: Preparation ofH₂N-G-G-G-G-G-Q-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) (DisulfideC⁹-C¹²) (SEQ ID NO: 70), Intermediate 43c

To intermediate 43b (44 mg, 0.028 mmol) in 0.9 mL of H₂O was added I₂(50 mM in AcOH, 1.1 mL 0.055 mmol) dropwise. The mixture was shaked atroom temperature overnight. LC/MS showed the reaction completed. To thereaction mixture was added several drops of 0.5 M of ascorbic acidsolution (MeOH/H₂O=1/1) until the color of the solution disappeared. Themixture was diluted with MeOH for HPLC purification. The purificationwas carried out by preparative HPLC (Sunfire™ Prep C18 OBD™ 30×50 mm 5um column ACN/H2O w/0.1% TFA 75 ml/min, 10-30% ACN 8 min gradient). Theproduct fraction was lyophilized to giveH₂N-G-G-G-G-G-Q-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) (disulfideC⁹-C¹²) (SEQ ID NO: 70), intermediate 43c as TFA salt (13 mg, 30%).LC/MS (QT2, ProductAnalysis-HRMS-Acidic, Waters Acquity UPLC BEH C18 1.7um 2.1×50 mm, 50° C., Eluent A: Water+0.1% Formic Acid, Eluent B:Acetonitrile+0.1% Formic Acid, gradient 2% to 98% B/A over 5.15 mins):Rentention time: 0.98 mins; MS [M+2]²⁺: observed: 1587.7993, calculated:1587.868.

Step 3: Sortase Conjugation of Fc-Sortase and Intermediate 43c 1)Chemoenzymatic Sortase Conjugation

On ice bath, to the Fc-Sortase (698 μl, 0.040 μmol, 3.15 mg/mL) in PBS(pH7.4) buffer solution was added the solution ofH₂N-G-G-G-G-G-Q-R-P-C*-L-S-C*-K-G-P-(D-Nle)-NH(Phenethyl) (disulfideC⁹-C¹²) (SEQ ID NO: 70) (64.1 μL, 2.018 μmoL, 50 mg/mL) in Tris-8.0buffer, followed by 520 μM of sortase A (78 μL, 0.040 μmoL) in 50 mMTris-Cl pH7.4, 150 mM NaCl. The mixture was shaked at room temperatureovernight. LC/MS showed the reaction completed.

2) Purification and Desalting

The above solution was flowed over a 5 mL HiTrap Mab Select SuRe column(GE Lifesciences #11-0034-95) at 4 mL/min on ATTA XPRESS. Example 43 waswashed on the column with 20 column volumes (CV) PBS+0.1% Triton 114 andeluted with 0.1M glycine, pH 2.7, neutralized with 1 M tris-HCl, pH 9and dialyzed versus PBS. The purified solution was desalted by usingZeba Sping Desalting Column, 5 mL (89891) to give 1.5 mL targetsolution, the average concentration was 0.598 mg/mL, and the recoveragewas 90%. LCMS (QT2, Protein_20-70 kDa_3 min, AcQuity ProSwift RP-3U4.6×50 mm, 1.0 mL/min, Eluent A: Water+0.1% Formic Acid, Eluent B:Acetonitrile+0.1% Formic Acid, gradient 2% to 98% B/A over 3 mins):R_(t)=1.55 minutes, MS [M+H] 58845.0000.

Sequence of the Example 43: (SEQ ID NO: 76) 1METDTLLLWV LLLWVPGSTG DKTHTCPPCP APEAAGGPSV FLFPPKPKDT 51LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY 101RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 151LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 201DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG 251GSLPETGGGGGQRPC*LSC*KGP(D-Nle)Phenethylaminewherein LSLSPGKGGG GSLPETGGGGG (SEQ ID NO: 77) represents the linker andQRPC*LSC*KGP(D-Nle)Phenethylamine (SEQ ID NO: 78) the polypeptide.

The polypeptides in the examples below have been found to have EC₅₀values in the range of about 0.01 nM to about 1100 nM for APJ receptorpotency. The polypeptides in the examples below have been found to havea plasma stability higher than 2 minutes, higher than 5 minutes, higherthan 10 minutes, higher than 20 minutes, higher than 50 minutes andhigher than 60 minutes.

It can be seen that the polypeptides of the invention are useful asagonist of the APJ receptor and therefore useful in the treatment ofdiseases and conditions responsive to the activation of the APJreceptor, such as the diseases disclosed herein.

Furthermore, half-life of these peptides can be further extended byforming a bioconjugate comprising a peptide or polypeptide according toany one of Formula I to IV with a half-life extending moiety, such asHuman serum Albumin or a Fc domain.

Having thus described exemplary embodiments of the present invention, itshould be noted by those of ordinary skill in the art that the withindisclosures are exemplary only and that various other alternatives,adaptations, and modifications may be made within the scope of thepresent invention. Accordingly, the present invention is not limited tothe specific embodiments as illustrated therein.

What is claimed is:
 1. A cyclic polypeptide having the following formulaI (SEQ ID NO: 1):X1-R-X3-X4-L-S-X7-X8-X9-X10-X11-X12-X13  I wherein: X1 is the N-terminusof the polypeptide and is either absent, Q, A or pE or X1 is selectedfrom C, c, hC, D-hC; wherein the side chain of C, c, hC or D-hC form adisulfide bond with the side chain of X7; X3 is P or X3 is selected fromC, c, hC and D-hC; wherein the side chain of C, c, hC or D-hC forms adisulfide bond with the side chain of X7; X4 is R; wherein only one ofX1 and X3 is a sulfur contain amino-acid selected from C, c, hC andD-hC; X7 is C, c, hC or D-hC; and the side chain of X7 forms a disulfidebond with the side chain of C, c, hC or D-hC of either X1 or X3; X8 is Kor F; X9 is G, A, a or absent; X10 is P or absent; X11 is D-Nle, Nle, Mor f; and X12 is absent or P, f, a, D-Nva or D-Abu; X13 is theC-terminus and is absent or is selected from (N-Me)F, F, f, a, y andNal; wherein: Nle is L-norleucine; D-Nle is D-norleucine; D-hC isD-homocysteine hC is L-homocysteine; Nal is L-naphathaline; D-Nva isD-norvaline; D-Abu is D-2-aminobutyric acid; pE is L-pyroglutamic acid;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.
 2. (canceled)
 3. The polypeptide ofclaim 1 having Formula III (SEQ ID NO: 4):

Wherein X1 is the N-terminus of the polypeptide and is selected from C,c, hC and D-hC; X7 is C, c, hC or D-hC; wherein the side chain of X7forms a disulfide bond with the side chain of X1; X8 is K or F; X9 is G,A, a or absent; X10 is P or absent; X11 is D-Nle, Nle, M or f; and X12is absent or is selected from P, f, a, D-Nva and D-Abu; X13 is theC-terminus and is absent or is selected from (N-Me)F, F, f, a, y andNal; or an amide, an ester or a salt of the polypeptide.
 4. Thepolypeptide of claim 1 having Formula IV (SEQ ID NO: 5):

wherein: X1 is the N-terminus of the polypeptide and is either absent,Q, A or pE; X3 is C, c, hC or D-hC; wherein the side chain of C, c, hCor D-hC; X7 is C, c, hC or D-hC; and the side chain of X7 form adisulfide bond with the side chain of C, c, hC or D-hC of X3; X8 is K orF; X9 is G, A, a or absent; X10 is P or absent; X11 is D-Nle, Nle, M orf; and X12 is absent or is selected from P, f, a, D-Nva and D-Abu; X13is the C-terminus and is absent or is selected from (N-Me)F, F, f, a, yand Nal; or an amide, an ester or a salt of the polypeptide.
 5. Thepolypeptide according claim 1 wherein X1 is pE; or an amide, an ester ora salt of the polypeptide.
 6. The polypeptide according to claim 1wherein X1 is absent; or an amide, an ester or a salt of thepolypeptide.
 7. The polypeptide according to claim 1 wherein theN-terminus is an amide; or a salt of the polypeptide.
 8. The polypeptideaccording to claim 7 wherein the N-terminus is an amide of Formula —NHRand R is Acetyl, benzoyl, phenacyl, succinyl, octanoyl,4-phenylbutanoyl, 4-Cl-Ph-(CH₂)₃C(O)—, or Ph-CH₂CH₂NHC(O)—; or a salt ofthe polypeptide.
 9. The polypeptide according to claim 1 wherein X13 isF or f; or an amide, an ester or a salt of the polypeptide.
 10. Thepolypeptide according to claim 1 wherein X13 is absent; or an amide, anester or a salt of the polypeptide.
 11. The polypeptide according toclaim 1 wherein X12 is absent; or an amide, and ester or a salt of thepolypeptide.
 12. The polypeptide according to claim 1 wherein theC-terminus is an amide; or a salt of the polypeptide.
 13. Thepolypeptide according to claim 12 wherein the C-terminus is an amide ofFormula —C(O)—R2 and R2 is —NH₂, —NH-Me, —NH—NHBn, or —NH—(CH₂)₂-Ph; ora salt of the polypeptide.
 14. The polypeptide according to claim 1wherein X8 is K; or an amide, an ester or a salt of the polypeptide. 15.The polypeptide according to claim 1 wherein X9 is G; or an amide, anester or a salt of the polypeptide.
 16. The polypeptide according toclaim 1 wherein X10 is P; or an amide, an ester or a salt of thepolypeptide.
 17. The polypeptide according to claim 1 wherein X11 is Nleor D-Nle; or an amide, an ester or a salt of the polypeptide.
 18. Thepolypeptide according to claim 1 selected from (SEQ ID Nos 19-45,respectively, in order of appearance):Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-F-OHAc-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NH(Phenethyl)Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-(N-Me)F-OHAc-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NH ₂Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-Nal-OHAc-C*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OHAc-hC*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OHAc-C*-R-P-R-L-S-C*-K-G-P-Nle-P-a-OHAc-C*-R-P-R-L-S-C*-K-G-P-Nle-P-NMe(Phenethyl)Ac-C*-R-P-R-L-S-C*-K-G-P-Nle-P-f-OHAc-C*-R-P-R-L-S-C*-K-G-P-Nle-NH(Phenethyl)Ac-c*-R-P-R-L-S-C*-K-G-P-Nle-P-F-OH Ac-c*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OHAc-c*-R-P-R-L-S-c*-K-G-P-Nle-P-F-OH Ac-C*-R-P-R-L-S-c*-K-G-P-Nle-P-F-OHAc-(D-hC)*-R-P-R-L-S-hC*-K-G-P-Nle-P-F-OHAc-(D-hC)*-R-P-R-L-S-(D-hC)*-K-G-P-Nle-P-F-OHAc-(D-hC)*-R-P-R-L-S-(hC)*-K-G-P-f-a-f-OHAc-c-R-P-R-L-S-(hC)-K-G-P-(D-Nle)-a-f-OHpE-R-C*-R-L-S-C*-K-G-P-Nle-P-F-OH pE-R-C*-R-L-S-C*-K-G-P-(D-Nle)-a-f-OHpE-R-C*-R-L-S-C*-F-G-P-(D-Nle)-a-f-OHpE-R-C*-R-L-S-C*-K-a-P-(D-Nle)-a-f-OHpE-R-C*-R-L-S-C*-K-A-P-(D-Nle)-a-f-OH pE-R-C*-R-L-S-C*-K-G-P-(D-Nle)-NH₂ pE-R-C*-R-L-S-(D-hC)*-K-G-P-f-a-f-OHpE-R-c*-R-L-S-C*-K-G-P-(D-Nle)-(D-abu)-f-OH

wherein the two amino acids labeled with “*” represent the amino acidsforming a disulfide; or an amide, an ester or a salt of the polypeptide.19. A bioconjugate or multimer thereof comprising: a. a peptide orpolypeptide according to claim 1, or an amide, an ester or a saltthereof, and b. a half-life extending moiety; wherein said peptide orpolypeptide and half-life extending moiety are covalently linked orfused, optionally via a linker.
 20. The bioconjugate or a multimerthereof, according to claim 19, wherein the half-life extending moietyis an IgG constant domain or fragment thereof or a human Serum Albumin.21-27. (canceled)
 28. The bioconjugate according to claim 19 wherein thehalf-life extending moiety is a fatty acid. 29-30. (canceled)
 31. Amethod of treating or preventing a disease or disorder responsive to theagonism of the APJ receptor, in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of apolypeptide or an amide, an ester or a salt thereof, according toclaim
 1. 32. The method of claim 31 wherein the disease or disorder isselected from acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.33-35. (canceled)
 36. A Combination comprising a therapeuticallyeffective amount of a polypeptide, an amide, an ester of a salt thereof,according to claim 1, and one or more therapeutically active co-agent.37. A combination according to claim 36 wherein the co-agent is selectedfrom inotropes, beta adrenergic receptor blockers, HMG-Co-A reductaseinhibitors, angiotensin II receptor antagonists, angiotensin convertingenzyme (ACE) Inhibitors, calcium channel blockers (CCB), endothelinantagonists, renin inhibitors, diuretics, ApoA-I mimics, anti-diabeticagents, obesity-reducing agents, aldosterone receptor blockers,endothelin receptor blockers, aldosterone synthase inhibitors (ASI), aCETP inhibitor, anti-coagulants, relaxin, BNP (nesiritide) and/or a NEPinhibitor.
 38. A pharmaceutical composition comprising a therapeuticallyeffective amount of a polypeptide, an amide, an ester of a salt thereof,according to claim 1, and one or more pharmaceutically acceptablecarriers.
 39. A pharmaceutical composition comprising a therapeuticallyeffective amount of a bioconjugate according to claim 19, and one ormore pharmaceutically acceptable carriers.
 40. A method of treating orpreventing a disease or disorder responsive to the agonism of the APJreceptor, in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a bioconjugate accordingto claim 19.